Sigma-2 receptor ligand drug conjugates as antitumor compounds, methods of synthesis and uses thereof

ABSTRACT

Methods and compositions for treating cancers such as pancreatic cancer and synovial sarcoma are disclosed. Compounds comprising a sigma-2 receptor-binding moiety and a ferroptosis-inducing moiety are described, such as the methanesulfonate salt of a compound of structural Formula IV, 
                         
, wherein n is an integer chosen from 1, 2, 3, 4, and 5, and R 2  is H or methyl. At least one described molecular species exhibits an IC 50  value below 5 μM against human pancreatic cancer cells in vitro. Administration of this species promoted shrinkage of pancreatic cancer tumors in a murine model system in vivo. It led to a 100% survival of experimental animals over a time course in which control therapies provided only 30% or 40% survival. Methods of synthesis of molecular species are also disclosed.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/111,842, filed Aug. 24, 2018, which is a continuation of U.S.application Ser. No. 15/301,188, filed Sep. 30, 2016, now U.S. Pat. No.10,087,175, issued Oct. 2, 2018, which claims the benefit ofPCT/US2015/023954, filed Apr. 1, 2015, which claims the benefit of U.S.Provisional Application No. 61/973,366, filed Apr. 1, 2014, all of whichare hereby incorporated by reference in their entireties for allpurposes.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

The invention was made with government support under CA163764 awarded bythe National Institute of Health. The government has certain rights inthe invention

FIELD

The present disclosure relates to sigma-2 receptor binding compounds andcompositions, and their application as pharmaceuticals for the treatmentof disease. More particularly, embodiments are related to sigma-2receptor ligand-drug conjugates, their synthesis and their use fortreating hyperproliferative diseases such as cancer.

DESCRIPTION OF RELATED ART

Pancreatic cancer is the fourth leading cause of cancer death, and isexpected to be the second leading cause in 2020. The five-year survivalrate for pancreatic cancer is only 5.8% with current treatment optionsand there is a desperate need for new therapies.

Sigma-2 receptors (S2R) are over-expressed in pancreatic ductaladenocarcinomas (PDAC) cells and have a high affinity for S2R ligands.S2R ligands localize to PDAC cells and are rapidly internalized by thecancer cells, eventually leading to apoptosis and cell death. S2Rligands also potentiate conventional anticancer chemotherapies andimprove survival in models of pancreatic adenocarcinoma.

S2R ligands linked to small molecule imaging tags have been used todemonstrate that S2R ligands preferentially bind to pancreaticadenocarcinomas, and can be used to visualize the S2R on cancer cells.Similarly. S2R ligands can be linked to proapoptotic peptides orpeptidomimetics, which can be selectively delivered into cancer cells.

Erastin, of structure:

is a drug with selectivity for killing cells with oncogenic K-rasmutations by mediating cell death by an iron-dependent, non-apoptoticprocess termed ferroptosis. However, this drug underperformed in initialclinical trials.

Erastin has been described in US Patent Application Publication2007/0161644 of Stockwell, B. R., as having cell killing properties thatare non-apoptotic. This reference also disclosed certain Erastinanalogs, such as Erastin A of structure

Erastin B of Structure

and des-methyl Erastin (designated “compound 21”) of structure

(also designated herein as SW V-27). The inventor in this applicationasserted that des-methyl Erastin has tumor cell-killing activitycomparable to that of Erastin. Dixon, S. J., et al., Cell 149:1060-1072, 2012 describes Erastin as mediating a nonapoptotic,iron-dependent oxidative cell death (“ferroptosis”). However, thesereferences do not describe compounds comprising a bicyclic moiety, andthe compounds described do not also mediate apoptotic cell death.

U.S. Pat. No. 8,143,222 to McDunn, J. E., et al, as well as Spitzer, D.et al., Cancer Res. 72: 201-209, 2012 and Hornick, J. R., et al.Molecular Cancer 9: 298, 2010 disclose compounds for beating cancer.Compounds disclosed in these references include molecules having atargeting moiety which binds sigma-2 receptor (Zeng, C., et al., CancerRes. 67: 6708-6716, 2007) and an apoptosis-inducing moiety such as aproapoptotic peptide. Disclosed compounds include a sigma-2 receptorligand such as an N-substituted 9-azabicyclo[3.3.1]nonan-3α-ylphenylcarbamate moiety of structure:

wherein R can be selected from the group consisting of a bond, a C₁-C₁₀alkyl, a C₁-C₁₀ alkylamine, a C₁-C₁₀alkylamide, a C₁-C₁₀heteroalkyl, aC₁-C₁₀aryl, a C₁-C₁₀heteroaryl, an ester and a hydrophilic polymer. Insome configurations, compounds of this patent include alkylaminederivatives of a bicyclic phenylcarbamate moiety, such as

(Designated SV119 when n=1) and SW43 of structure

Although some compounds disclosed in these references are described ashaving tumor cell killing activity, none of them are disclosed tomediate iron-dependent oxidative cell death (ferroptosis).

SUMMARY

Accordingly, the inventors herein disclose new compositions andcompounds and methods of their synthesis, and methods for treatinghyperproliferative disorders, including various cancers.

In various embodiments, the present teachings include compounds ofstructural Formula I

and salts thereof, wherein: W can be an aryl group such as a C₅-C₁₀ arylor a C₅-C₁₀ heteroaryl, any of which can be substituted; X can be alinking moiety such as, but not limited to a linear alkyl chain; and Ycan be a ferroptosis-inducing moiety such as erastin, an erastin analogsuch as erastin-A, erastin-B, desmethyl-erastin or an erastin mimetic.

In various embodiments, the present teachings include compositionscomprising a compound of Formula I and a pharmaceutically acceptablecarrier, adjuvant, or vehicle.

In various embodiments, the present teachings include methods oftreating a hyperproliferative disorder in a subject in need thereof,comprising the step of administering to the subject a compound ofFormula I.

In various embodiments, the present teachings include methods oftreating a hyperproliferative disorder in a subject in need thereof,comprising the sequential or co-administration of a compound of FormulaI or a pharmaceutically acceptable salt thereof, and another therapeuticagent.

In various embodiments, the present teachings include compounds ofFormula I for use in human therapy.

In various embodiments, the present teachings include compounds of anyof Formula I for use in treating a hyperproliferative disorder.

In various embodiments, the present teachings include use of a compoundof Formula I for the manufacture of a medicament to treat ahyperproliferative disorder.

In various embodiments, the present teachings include compounds or asalt thereof, of structure

wherein n is an integer from 1 to 5 and R₁ can be H or methyl. In someconfigurations, compounds or salts of these embodiments includecompounds and salts wherein n can be 1 and R₁ can be H. In someconfigurations, compounds or salts of these embodiments includecompounds and salts wherein n can be 5 and R₁ can be H. In variousconfigurations, a salt of a compound of these embodiments can be anoxalate salt.

In various embodiments, these compounds and salts thereof can be used inmethods of treating cancers. In various configurations, these methodscan comprise administering to a subject in need thereof atherapeutically effective amount any of these compounds or saltsthereof. In various configurations, a cancer that can be treated withany of these compounds or salts thereof can be any cancer, such as,without limitation, a pancreatic cancer or a synovial sarcoma.

In various embodiments, these compounds and salts thereof can besynthesized by methods disclosed herein. In some configurations, thesemethods can comprise reacting a compound of structure

with a compound of structure

wherein n can be an integer from 1 to 5 and R, can be methyl or H. Insome configurations, n can be 1 and R₁ can be an H. In someconfigurations, n can be 5 and R₁ can be an H.

In some configurations, the present teachings include these compounds orsalts thereof for use in the treatment of a cancer. In some aspects, thecancer can be, without limitation, a pancreatic cancer or a synovialsarcoma.

In some configurations, the present teachings include use of thesecompounds or salts thereof for the manufacture of a medicament for thetreatment of cancer. In some aspects, the cancer can be, withoutlimitation, a pancreatic cancer or a synovial sarcoma.

In various embodiments, the present teachings include methods ofsynthesizing a compound of Formula IV:

comprising the step of reacting a compound of structural Formula V:

with a compound of structural Formula VI:

wherein n is an integer from 1 to 5; and R² can be H or methyl.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates Erastin and des-methyl Erastin (SW V-27).

FIG. 2A-C illustrate synthesis of compounds SW V-49s and SW V-50s of thepresent teachings.

FIG. 3 illustrates competitive internalization inhibition of thefluorescent sigma-2 ligand SW 120 with SW V-49s by Pane-1 cells.

FIG. 4A-F illustrate viability assays of SW V-49s on various human (A-D)and murine (E) pancreatic cancer cell lines in vitro.

FIG. 5A-B illustrate increased apoptotic cell death induced by SW V-49sassayed after treatment for 24 hr. (A) or 7 hr. (B).

FIG. 6 illustrates ferroproptotic cell death induced by SW V-49s.

FIG. 7 illustrates decrease in tumor size in pancreatic cancer followingSW V-49s administration in a murine model system.

FIG. 8 illustrates 100% survival of pancreatic cancer following SW V-49sadministration in a murine model system.

FIG. 9 illustrates the cell killing characteristics of SW V-49s andcontrols.

FIG. 10 illustrates a cell viability assay of cells treated withErastin.

FIG. 11A-B illustrate that SW V-49s inhibits cystine uptake resulting inROS generation.

FIG. 12A-C illustrate that SW V-49s treatment induces intrinsicapoptotic pathway.

FIG. 13 illustrate that SW V-49s induces apoptotic and ROS dependentcell death.

FIG. 14A-E illustrate that SW V49s reduces tumor growth and enhancessurvival.

DETAILED DESCRIPTION Abbreviations and Definitions

To facilitate understanding of the disclosure, a number of terms andabbreviations as used herein are defined below as follows:

When introducing elements of the present disclosure or the preferredembodiment(s) thereof, the articles “a”. “an”, “the” and “said” areintended to mean that there are one or more of the elements.

Chemical species and moieties are named in accordance with Naming andIndexing of Chemical Substances for Chemical Abstracts™ 2007 Edition.American Chemical Society, 2008, except as specified below.

The term “lower.” as used herein, alone or in a combination, where nototherwise specifically defined, means containing from 1 up to andincluding 6 carbon atoms.

The term “lower aryl.” as used herein, alone or in combination, meansphenyl or naphthyl, either of which can be optionally substituted asprovided.

The term “lower heteroaryl,” as used herein, alone or in combination,means either 1) monocyclic heteroaryl comprising five or six ringmembers, of which between one and four the members can be heteroatomsselected from the group consisting of O, S, and N, or 2) bicyclicheteroaryl, wherein each of the fused rings comprises five or six ringmembers, comprising between them one to four heteroatoms selected fromthe group consisting of O, S, and N.

The term “lower cycloalkyl,” as used herein, alone or in combination,means a monocyclic cycloalkyl having between three and six ring members.Lower cycloalkyls can be unsaturated. Examples of lower cycloalkylinclude cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

The term “lower heterocycloalkyl.” as used herein, alone or incombination, means a monocyclic heterocycloalkyl having between threeand six ring members, of which between one and four can be heteroatomsselected from the group consisting of O, S, and N. Examples of lowerheterocycloalkyls include pyrrolidinyl, imidazolidinyl, pyrazolidinyl,piperidinyl, piperazinyl, and morpholinyl. Lower heterocycloalkyls canbe unsaturated.

The term “lower amino,” as used herein, alone or in combination, refersto —NRR′, wherein R and R′ are independently selected from the groupconsisting of hydrogen, alkyl, and lower heteroalkyl, any of which canbe optionally substituted. Additionally, the R and R′ of a lower aminogroup can combine to form a five- or six-membered heterocycloalkyl,either of which can be optionally substituted.

The terms “oxy” or “oxa.” as used herein, alone or in combination, referto —O—.

The term “oxo,” as used herein, alone or in combination, refers to ═O.

The term “perhaloalkoxy” refers to an alkoxy group where all of thehydrogen atoms are replaced by halogen atoms.

The term “perhaloalkyl” as used herein, alone or in combination, refersto an alkyl group where all of the hydrogen atoms are replaced byhalogen atoms.

The terms “sulfonate”, “sulfonic acid,” and “sulfonic.” as used herein,alone or in combination, refer to the —SO₃H group and its anion as thesulfonic acid as used in salt formation.

The term “sulfanyl,” as used herein, alone or in combination, refers to—S—.

The term “sulfinyl.” as used herein, alone or in combination, refers to—S(O)—.

The term “N-sulfonamide” refers to a RS(═O)₂NR′-group with R and R′ asdefined herein.

The term “S-sulfonamido” refers to a —S(═O)₂NRR′, group, with R and R′as defined herein.

The terms “thia” and “thio,” as used herein, alone or in combination,refer to a —S— group or an ether wherein the oxygen is replaced withsulfur. The oxidized derivatives of the thio group, namely sulfinyl andsulfonyl, are included in the definition of thia and thio.

The term “thiocarbonyl,” as used herein, when alone includes thioformyl—C(S)H and in combination is a —C(S)— group.

The term “N-thiocarbamyl” refers to an ROC(S)NR′— group, with R and R′as defined herein.

The term “O-thiocabamyl” refers to a —OC(S)NRR′, group with R and R′ asdefined herein.

The term “thiocyanato” refers to a —CNS group.

The term “trihalomethanesulfonamido” refers to a X₃CS(O)₂NR— group withX is a halogen and R as defined herein.

The term “trihalomethanesulfonyl” refers to a X₃CS(O)₂— group where X isa halogen.

The term “trihalomethoxy” refers to a X₃CO— group where X is a halogen.

The term “trisubstituted silyl.” as used herein, alone or incombination, refers to a silicone group substituted at its three freevalences with groups as listed herein under the definition ofsubstituted amino. Examples include trimethysilyl,tert-butyidimethylsilyl, triphenylsilyl and the like.

Any definition herein can be used in combination with any otherdefinition to describe a composite structural group. By convention, thetrailing element of any such definition is that which attaches to theparent moiety. For example, the composite group alkylamido wouldrepresent an alkyl group attached to the parent molecule through anamido group, and the term alkoxyalkyl would represent an alkoxy groupattached to the parent molecule through an alkyl group.

The term “optionally substituted” means the anteceding group can besubstituted or unsubstituted. When substituted, the substituents of an“optionally substituted” group can include, without limitation, one ormore substituents independently selected from the following groups or aparticular designated set of groups, alone or in combination: alkyl,lower alkenyl, lower alkynyl, lower alkanoyl, lower heteroalkyl, lowerheterocycloalkyl, lower haloalkyl, lower haloalkenyl, lower haloalkynyl,lower perhaloalkyl, lower perhaloalkoxy, lower cycloalkyl, phenyl, aryl,aryloxy, lower alkoxy, lower haloalkoxy, oxo, lower acyloxy, carbonyl,carboxyl, alkylcarbonyl, lower carboxyester, lower carboxamido, cyano,hydrogen, halogen, hydroxy, amino, alkylamino, arylamino, amido, nitro,thiol, alkylthio, lower haloalkylthio, lower perhaloalkylthio, arylthio,sulfonate, sulfonic acid, trisubstituted silyl, N₃, SH, SCH₃, C(O)CH₃,CO₂CH₃, CO₂H, pyridinyl, thiophene, furanyl, lower carbamate, and lowerurea. Two substituents can be joined together to form a fused five-,six-, or seven-membered carbocyclic or heterocyclic ring consisting ofzero to three heteroatoms, for example forming methylenedioxy orethylenedioxy. An optionally substituted group can be unsubstituted(e.g., —CH₂CH₃), fully substituted (e.g., —CF₂CF₃), monosubstituted(e.g., —CH₂CH₂F) or substituted at a level anywhere in-between fullysubstituted and monosubstituted (e.g., —CH₂CF₃). Where substituents arerecited without qualification as to substitution, both substituted andunsubstituted forms are encompassed. Where a substituent is qualified as“substituted.” the substituted form is specifically intended.Additionally, different sets of optional substituents to a particularmoiety can be defined as needed; in these cases, the optionalsubstitution will be as defined, often immediately following the phrase,“optionally substituted with.”

The term R or the term R′ appearing by itself and without a numberdesignation, unless otherwise defined, refers to a moiety selected fromthe group consisting of hydrogen, alkyl, cycloalkyl, heteroalkyl, aryl,heteroaryl and heterocycloalkyl, any of which can be optionallysubstituted. Such R and R′ groups should be understood to be optionallysubstituted as defined herein. Whether an R group has a numberdesignation or not, every R group, including R. R′ and Rn wherein n isan integer, every substituent, and every term should be understood to beindependent of every other in terms of selection from a group. Shouldany variable, substituent, or term (e.g. aryl, heterocycle, R, etc.)occur more than one time in a formula or generic structure, itsdefinition at each occurrence is independent of the definition at everyother occurrence. Those of skill in the art will further recognize thatcertain groups can be attached to a parent molecule or can occupy aposition in a chain of elements from either end as written. Thus, by wayof example only, an unsymmetrical group such as —C(O)N(R)— can beattached to the parent moiety at either the carbon or the nitrogen.

Asymmetric centers exist in the compounds disclosed herein. Individualstereoisomers of compounds can be prepared synthetically fromcommercially available starting materials which contain chiral centersor by preparation of mixtures of enantiomeric products followed byseparation such as conversion to a mixture of diastereomers followed byseparation or recrystallization, chromatographic techniques, directseparation of enantiomers on chiral chromatographic columns, or anyother appropriate method known in the art. Starting compounds ofparticular stereochemistry are either commercially available or can bemade and resolved by techniques known in the art. Additionally, thecompounds disclosed herein can exist as geometric isomers. Additionally,compounds can exist as tautomers; all tautomeric isomers are provided bythis disclosure. Additionally, the compounds disclosed herein can existin unsolvated as well as solvated forms with pharmaceutically acceptablesolvents such as water, ethanol, and the like. In general, the solvatedforms are considered equivalent to the unsolvated forms.

The term “bond” refers to a covalent linkage between two atoms, or twomoieties when the atoms joined by the bond are considered part of largersubstructure. A bond can be single, double, or triple unless otherwisespecified. A dashed line between two atoms in a drawing of a moleculeindicates that an additional bond can be present or absent at thatposition.

The term “disease” as used herein is synonymous, and is usedinterchangeably with, the terms “disorder,” and “condition” (as inmedical condition), in that all reflect an abnormal condition of thehuman or animal body or of one of its parts that impairs normalfunctioning, is typically manifested by distinguishing signs andsymptoms, and causes the human or animal to have a reduced duration orquality of life.

The term “combination therapy” means the administration of two or moretherapeutic agents to treat a therapeutic condition or disorderdescribed in the present disclosure. Such administration encompassesco-administration of these therapeutic agents in a substantiallysimultaneous manner, such as in a single capsule having a fixed ratio ofactive ingredients or in multiple, separate capsules for each activeingredient. In addition, such administration also encompasses use ofeach type of therapeutic agent in a sequential manner.

The phrase “therapeutically effective” is intended to qualify the amountof active ingredients used in the treatment of a disease or disorder oron the effecting of a clinical endpoint.

The term “therapeutically acceptable” refers to those compounds (orsalts, prodrugs, tautomers, zwitterionic forms, etc.) which are suitablefor use in contact with the tissues of patients without undue toxicity,irritation, and allergic response, are commensurate with a reasonablebenefit/risk ratio, and are effective for their intended use.

In the present disclosure, the term “radiation” means ionizing radiationcomprising particles or photons that have sufficient energy or canproduce sufficient energy via nuclear interactions to produce ionization(gain or loss of electrons). An exemplary and preferred ionizingradiation is an x-radiation. Means for delivering x-radiation to atarget tissue or cell are well known in the art. The amount of ionizingradiation needed in a given cell generally depends on the nature of thatcell. Means for determining an effective amount of radiation are wellknown in the art. Used herein, the term “an effective dose” of ionizingradiation means a dose of ionizing radiation that produces an increasein cell damage or death.

The term “radiation therapy” refers to the use of electromagnetic orparticulate radiation in the treatment of neoplasia and includes the useof ionizing and non-ionizing radiation.

The compounds disclosed herein can exist as therapeutically acceptablesalts. The present disclosure includes compounds listed above in theform of salts, including acid addition salts. Suitable salts includethose formed with both organic and inorganic acids. Such acid additionsalts will normally be pharmaceutically acceptable. However, salts ofnon-pharmaceutically acceptable salts can be of utility in thepreparation and purification of the compound in question. Basic additionsalts can also be formed and be pharmaceutically acceptable. For a morecomplete discussion of the preparation and selection of salts, refer toPharmaceutical Salts: Properties, Selection, and Use (Stahl, P.Heinrich. Wiley-VCHA, Zurich. Switzerland, 2002).

The term “therapeutically acceptable salt.” as used herein, representssalts or zwitterionic forms of the compounds disclosed herein which arewater or oil-soluble or dispersible and therapeutically acceptable asdefined herein. The salts can be prepared during the final isolation andpurification of the compounds or separately by reacting the appropriatecompound in the form of the free base with a suitable acid.Representative acid addition salts include acetate, adipate, alginate,L-ascorbate, aspartate, benzoate, benzenesulfonate (besylate),bisulfate, butyrate, camphorate, camphorsulfonate, citrate, digluconate,formate, fumarate, gentisate, glutarate, glycerophosphate, glycolate,hemisulfate, heptanoate, hexanoate, hippurate, hydrochloride,hydrobromide, hydroiodide, 2-hydroxyethansulfonate (isethionate),lactate, maleate, malonate. DL-mandelate, mesitylenesulfonate,methanesulfonate, naphthylenesulfonate, nicotinate,2-naphtlialenesulfonate, oxalate, pamoate, pectinate, persulfate,3-phenylproprionate, phosphonate, picrate, pivalate, propionate,pyroglutamate, succinate, sulfonate, tartrate, L-tartrate,trichloroacetate, trifluoroacetate, phosphate, glutamate, bicarbonate,para-toluenesulfonate (p-tosylate), and undecanoate. Also, basic groupsin the compounds disclosed herein can be quaternized with methyl, ethyl,propyl, and butyl chlorides, bromides, and iodides; dimethyl, diethyl,dibutyl, and diamyl sulfates, decyl, lauryl, myristyl, and sterylchlorides, bromides, and iodides; and benzyl and phenethyl bromides.Examples of acids which can be employed to form therapeuticallyacceptable addition salts include inorganic acids such as hydrochloric,hydrobromic, sulfuric, and phosphoric, and organic acids such as oxalic,maleic, succinic, and citric. Salts can also be formed by coordinationof the compounds with an alkali metal or alkaline earth ion. Hence, thepresent disclosure contemplates sodium, potassium, magnesium, andcalcium salts of the compounds disclosed herein, and the like.

Basic addition salts can be prepared during the final isolation andpurification of the compounds by reacting a carboxy group with asuitable base such as the hydroxide, carbonate, or bicarbonate of ametal cation or with ammonia or an organic primary, secondary, ortertiary amine. The cations of therapeutically acceptable salts includelithium, sodium, potassium, calcium, magnesium, and aluminum, as well asnontoxic quaternary amine cations such as ammonium, tetramethylammonium,tetraethylammonium, methylamine, dimethylamine, trimethylamine,triethylamine, diethylamine, ethylamine, tributylamine, pyridine,N,N-dimethylaniline, N-methylpiperidine, N-methylmorpholine,dicyclohexylamine, procaine, dibenzylamine, N,N-dibenzylphenethylamine,1-ephenamine, and N,N′-dibenzylethylenediamine. Other representativeorganic amines useful for the formation of base addition salts includeethylenediamine, ethanolamine, diethanolamine, piperidine, andpiperazine.

Compounds

The present disclosure provides a compound of structural Formula I

or a sail thereof, wherein: W is chosen from optionally substitutedC₅-C₁₀ aryl and C₅-C₁₀ heteroaryl; X is a linking moiety; and Y is aferroptosis-inducing moiety chosen from erastin, an erastin analog suchas erastin-A, erastin-B, or desmethyl-erastin or a simplified syntheticerastin mimetic.

In some configurations. W can be a C₅-C₁₀ aryl.

In some configurations, W can be a substituted C₅-C₁₀ aryl.

In some configurations, W can be a sigma-2 receptor ligand.

In some configurations, the linking moiety can comprise a C₁-C₁₂ linearchain.

In some configurations, the linking moiety can further comprise 1 ormore heteroatoms. In some configurations, each of the 1 or moreheteroatoms can be independently selected from the group consisting ofan oxygen, a sulfur, and a nitrogen.

In some configurations, the linking moiety can have a structure

wherein

is a bond, and n is an integer from 1 to 10, i.e., 1, 2, 3, 4, 5, 6, 7,8, 9 or 10, 9. In some configurations, n=1. In some configurations, n=5.

In some configurations, the ferroptosis-inducing moiety of a compound ofthe structural Formula I can be selected from the group consisting oferastin, erastin-A, erastin-B and desmethyl-erastin.

In some configurations, the ferroptosis-inducing moiety of a compound ofthe structural Formula I can be erastin.

In some embodiments, the present teachings include compounds of FormulaII

or a salt thereof, wherein: W is chosen from optionally substitutedC₅-C₁₀ aryl and C₅-C₁₀ heteroaryl; X is a linking moiety; and R² ischosen from hydrogen and methyl.

In some embodiments, the present teachings include compounds of FormulaIII:

or a salt thereof, wherein: each of Z¹ and Z³ is independently selectedfrom the group consisting of a bond, C₁-C₁₀ alkyl. C₁-C₁₀ alkenyl,C₁-C₁₀ alkynyl. C₅-C₁₀ aryl, C₅-C₁₀ heteroaryl, arylalkyl,heteroarylalkyl, alkylaryl, alkylheteroaryl, heterocycloalkylalkyl,alkylheterocycloalkyl, cycloalkylalkyl, alkylcycloalkyl,—(CH₂)_(a)(OCH₂CH₂)_(b)(CH₂)_(c)—, —(CH₂)_(a)(CH₂CH₂O)(CH₂)_(c)—,—(CH₂)_(a)O(CH₂)_(c)—, —(CH₂)_(a)S(CH₂)_(c)—, —(CH₂)₃S(O)₂(CH₂)_(c)—,—(CH₂)_(a)S(O)(CH₂)_(c)—, —(CH₂)_(a)N(R)(CH₂)_(c)—,—(CH₂)_(a)N(R′)C(O)(CH₂)_(c), —(CH₂)_(a)(C(O)N(R¹)(CH₂)_(c)—,—(CH₂)_(a)N(R¹)C(O)N(R¹)(CH₂)_(c)—, —(CH₂)_(a)S(O)₂N(R¹)(CH₂)_(c)—,—(CH₂)_(a)N(R)S(O)₂(CH₂)_(c)—, Z² is chosen from C₁-C₁₀ alkyl, C₁-C₁₀alkenyl, C₁-C₁₀ alkynyl, C₅-C₁₀ aryl, C₅-C₁₀ heteroaryl, arylalkyl,heteroarylalkyl, alkylaryl, alkylheteroaryl, heterocycloalkylalkyl,alkylheterocycloalkyl, cycloalkylalkyl, alkylcycloalkyl,—(CH₂)_(a)(OCH₂CH₂)_(b)(CH₂)_(c)—, and—(CH₂)_(a)(CH₂CH₂O)_(b)(CH₂)_(c)—, —(CH₂)_(a)O(CH₂)_(c)—,—(CH₂)_(a)S(CH₂)_(c)—, —(CH₂)_(a)S(O)₂(CH₂)_(c)—, —(CH₂)_(a)S(O)(CH₂)_(c)—, —(CH₂)_(a)N(R¹)(CH₂)_(c)—, —(CH₂)_(a)N(R′)C(O)(CH₂)_(c)—,—(CH₂)_(a)C(O)N(R′)(CH₂)_(c)—, —(CH₂)_(a)N(R¹)C(O)N(R¹)(CH₂)_(c)—,—(CH₂)_(a)S(O)₂N(R¹)(CH₂)_(c)—, —(CH₂)_(a)N(R)S(O)₂(CH₂)_(c)—; whereineach of Z¹, Z², and Z³ can be optionally substituted with one or moregroups chosen from halo, oxo, and C₁-C₁₀ alkyl; each R¹ is independentlychosen from hydrogen, C₁-C₁₀ alkyl, and C₁-C₁₀ acyl; each a and c is aninteger independently chosen from 0, 1, 2, 3, and 4; each b is aninteger independently chosen from 1, 2, 3, 4, 5, and 6; and Y is aferroptosis-inducing moiety chosen from erastin, an erastin analog suchas erastin-A, erastin-B, or desmethyl-erastin or a simplified syntheticerastin mimetic.

In certain embodiments, a compound of the present teachings hasstructural Formula IV:

or a sail thereof, wherein: n is an integer chosen from 1, 2, 3, 4, and5: and R² can be selected from hydrogen and methyl.

In some configurations, n can be 1; and R² can be hydrogen.

In some configurations, n can be 5; and R² can be hydrogen.

In some configurations, the salt can be an oxalate salt.

In some configurations, the compound can be chosen from compounds 1-24as disclosed herein.

Pharmaceutical Compositions

While compounds of the present teachings can be administered as the rawchemical, it is also possible to present them as a pharmaceuticalformulations. Accordingly, provided herein are pharmaceuticalformulations which comprise one or more compounds of the presentteachings, or one or more pharmaceutically acceptable salts, esters,prodrugs, amides, or solvates thereof, together with one or morepharmaceutically acceptable carriers thereof and optionally one or moreother therapeutic ingredients. The carrier(s) must be “acceptable” inthe sense of being compatible with the other ingredients of theformulation and not deleterious to the recipient thereof. Properformulation is dependent upon the route of administration chosen. Any ofthe well-known techniques, carriers, and excipients can be used assuitable and as understood in the art; e.g., in Remington'sPharmaceutical Sciences. The pharmaceutical compositions disclosedherein can be manufactured in any manner known in the art, e.g., bymeans of conventional mixing, dissolving, granulating, dragee-making,levigating, emulsifying, encapsulating, entrapping or compressionprocesses.

The formulations include those suitable for oral, parenteral (includingsubcutaneous, intradermal, intramuscular, intravenous, intraarticular,and intramedullary), intraperitoneal, transmucosal, transdermal, rectaland topical (including dermal, buccal, sublingual and intraocular)administration although the most suitable route can depend upon forexample the condition and disorder of the recipient. The formulationscan conveniently be presented in unit dosage form and can be prepared byany of the methods well known in the art of pharmacy. Typically, thesemethods include the step of bringing into association a compound of thesubject disclosure or a pharmaceutically acceptable salt, ester, amide,prodrug or solvate thereof (“active ingredient”) with the carrier whichconstitutes one or more accessory ingredients. In general, theformulations are prepared by uniformly and intimately bringing intoassociation the active ingredient with liquid carriers or finely dividedsolid carriers or both and then, if necessary, shaping the product intothe desired formulation.

Compounds described herein can be administered as follows:

Oral Administration

The compounds of the present teachings can be administered orally,including swallowing, so the compound enters the gastrointestinal tract,or is absorbed into the blood stream directly from the mouth, includingsublingual or buccal administration.

Suitable compositions for oral administration include solid formulationssuch as tablets, pills, cachets, lozenges and hard or soft capsules,which can contain liquids, gels, powders, or granules.

In a tablet or capsule dosage form the amount of drug present can befrom about 0.05% to about 95% by weight, more typically from about 2% toabout 50% by weight of the dosage form.

In addition, tablets or capsules can contain a disintegrant, comprisingfrom about 0.5% to about 35% by weight, more typically from about 2% toabout 25% of the dosage form. Examples of disintegrates include methylcellulose, sodium or calcium carboxymethyl cellulose, croscarmellosesodium, polyvinylpyrrolidone, hydroxy propyl cellulose, starch and thelike.

Suitable binders, for use in a tablet, include gelatin, polyethyleneglycol, sugars, gums, starch, hydroxypropyl cellulose and the like.Suitable diluents, for use in a tablet, include mannitol, xylitol,lactose, dextrose, sucrose, sorbitol and starch.

Suitable surface active agents and glidants, for use in a tablet orcapsule, can be present in amounts from about 0.1% to about 3% byweight, and include polysorbate 80, sodium dodecyl sulfate, talc andsilicon dioxide.

Suitable lubricants, for use in a tablet or capsule, can be present inamounts from about 0.1% to about 5% by weight, and include calcium, zincor magnesium stearate, sodium stearyl fumarate and the like.

Tablets can be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets can be prepared bycompressing in a suitable machine the active ingredient in afree-flowing form such as a powder or granules, optionally mixed withbinders, inert diluents, or lubricating, surface active or dispersingagents. Molded tablets can be made by molding in a suitable machine amixture of the powdered compound moistened with a liquid diluent. Dyesor pigments can be added to tablets for identification or tocharacterize different combinations of active compound doses.

Liquid formulations can include emulsions, solutions, syrups, elixirsand suspensions, which can be used in soft or hard capsules. Suchformulations can include a pharmaceutically acceptable carrier, forexample, water, ethanol, polyethylene glycol, cellulose, or an oil. Theformulation can also include one or more emulsifying agents and/orsuspending agents.

Compositions for oral administration can be formulated as immediate ormodified release, including delayed or sustained release, optionallywith enteric coating.

In another embodiment, a pharmaceutical composition comprises atherapeutically effective amount of a compound of Formula (I) or apharmaceutically acceptable salt thereof, and a pharmaceuticallyacceptable carrier.

Parenteral Administration

Compounds of the present teachings can be administered directly into theblood stream, muscle, or internal organs by injection, e.g., by bolusinjection or continuous infusion. Suitable means for parenteraladministration include intravenous, intra-muscular, subcutaneousintraarterial, intraperitoneal, intrathecal, intracranial, and the like.Suitable devices for parenteral administration include injectors(including needle und needle-free injectors) and infusion methods. Theformulations can be presented in unit-dose or multi-dose containers, forexample sealed ampoules and vials.

Most parenteral formulations are aqueous solutions containingexcipients, including salts, buffering, suspending, stabilizing and/ordispersing agents, antioxidants, bacteriostats preservatives, andsolutes which render the formulation isotonic with the blood of theintended recipient, and carbohydrates.

Parenteral formulations can also be prepared in a dehydrated form (e.g.,by lyophilization) or as sterile non-aqueous solutions. Theseformulations can be used with a suitable vehicle, such as sterile water.Solubility-enhancing agents can also be used in preparation ofparenteral solutions.

Compositions for parenteral administration can be formulated asimmediate or modified release, including delayed or sustained release.Compounds can also be formulated as depot preparations. Such long actingformulations can be administered by implantation (for examplesubcutaneously or intramuscularly) or by intramuscular injection. Thus,for example, the compounds can be formulated with suitable polymeric orhydrophobic materials (for example as an emulsion in an acceptable oil)or ion exchange resins, or as sparingly soluble derivatives, forexample, as a sparingly soluble salt.

Topical Administration

In some configurations, compounds of the present teachings can beadministered topically (for example to the skin, mucous membranes, ear,nose, or eye) or transdermally. Formulations for topical administrationcan include, but are not limited to, lotions, solutions, creams, gels,hydrogels, ointments, foams, implants, patches and the like. Carriersthat are pharmaceutically acceptable for topical administrationformulations can include water, alcohol, mineral oil, glycerin,polyethylene glycol and the like. Topical administration can also beperformed by, for example, electroporation, iontophoresis, phonophoresisand the like.

Typically, the active ingredient for topical administration can comprisefrom 0.001% to 10% w/w (by weight) of the formulation. In certainembodiments, the active ingredient can comprise as much as 10% w/w: lessthan 5% w/w; from 2% w/w to 5% w/w; or from 0.1% to 1% w/w of theformulation.

Compositions for topical administration can be formulated as immediateor modified release, including delayed or sustained release.

Rectal, Buccal, and Sublingual Administration

Suppositories for rectal administration of the compounds of the presentteachings can be prepared by mixing the active agent with a suitablenon-irritating excipient such as cocoa butter, synthetic mono-, di- ortriglycerides, fatty acids, or polyethylene glycols which are solid atordinary temperatures but liquid at the rectal temperature, and whichwill therefore melt in the rectum and release the drug.

For buccal or sublingual administration, the compositions can take theform of tablets, lozenges, pastilles, or gels formulated in conventionalmanner. Such compositions can comprise the active ingredient in aflavored basis such as sucrose and acacia or tragacanth.

Administration by Inhalation

For administration by inhalation, in some configurations, compounds ofthe present teachings can be delivered from an insufflator, nebulizerpressurized packs or other convenient means of delivering an aerosolspray or powder. Pressurized packs can comprise a suitable propellantsuch as dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In thecase of a pressurized aerosol, the dosage unit can be determined byproviding a valve to deliver a metered amount. Alternatively, foradministration by inhalation or insufflation, the compounds according tothe disclosure can take the form of a dry powder composition, forexample a powder mix of the compound and a suitable powder base such aslactose or starch. The powder composition can be presented in unitdosage form, in for example, capsules, cartridges, gelatin or blisterpacks from which the powder can be administered with the aid of aninhalator or insufflator.

Other earner materials and modes of administration known in thepharmaceutical art can also be used. Pharmaceutical compositions of theteachings can be prepared by any of the well-known techniques ofpharmacy, such as effective formulation and administration procedures.Preferred unit dosage formulations are those containing an effectivedose, as herein recited, or an appropriate fraction thereof, of theactive ingredient. The precise amount of compound administered to apatient will be the responsibility of the attendant physician. Thespecific dose level for any particular patient will depend upon avariety of factors including the activity of the specific compoundemployed, the age, body weight, general health, sex, diets, time ofadministration, route of administration, rate of excretion, drugcombination, the precise disorder being treated, and the severity of theindication or condition being treated. In addition, the route ofadministration can vary depending on the condition and its severity. Theabove considerations concerning effective formulations andadministration procedures are well known in the art and are described instandard textbooks. Formulation of drugs is discussed in, for example.Hoover. John E., Remington's Pharmaceutical Sciences, Mack PublishingCo., Easton, Pa., 1975; Liberman, et al., Eds., Pharmaceutical DosageForms. Marcel Decker, New York. N.Y., 1980; and Kibbe, et al., Eds.,Handbook of Pharmaceutical Excipients (3^(rd) Ed.). AmericanPharmaceutical Association, Washington, 1999.

Methods of Treatment

The present disclosure provides compounds and pharmaceuticalcompositions feature a targeting moiety which binds the sigma-2 receptorand can thus be useful in the treatment or prevention of disordersassociated with cells that express the sigma-2 receptor and include, butare not limited to cancer.

Cancer

In some embodiments, the compounds and pharmaceutical compositions ofthe present disclosure can be useful in the treatment or prevention ofcancer.

In certain embodiments, the cancer can be chosen from adenocarcinoma,adult T-cell leukemia/lymphoma, bladder cancer, blastoma, bone cancer,breast cancer, brain cancer, carcinoma, myeloid sarcoma, cervicalcancer, colorectal cancer, esophageal cancer, gastrointestinal cancer,glioblastoma multiforme, glioma, gallbladder cancer, gastric cancer,head and neck cancer. Hodgkin's lymphoma, non-Hodgkin's lymphoma,intestinal cancer, kidney cancer, laryngeal cancer, leukemia, lungcancer, lymphoma, liver cancer, small cell lung cancer, non-small celllung cancer, mesothelioma, multiple myeloma, ocular cancer, optic nervetumor, oral cancer, ovarian cancer, pituitary tumor, primary centralnervous system lymphoma, prostate cancer, pancreatic cancer, pharyngealcancer, renal cell carcinoma, rectal cancer, sarcoma, skin cancer,spinal tumor, small intestine cancer, stomach cancer, synovial sarcoma.T-cell lymphoma, testicular cancer, thyroid cancer, throat cancer,urogenital cancer, urothelial carcinoma, uterine cancer, vaginal cancer,or Wilms' tumor.

In particular embodiments, the cancer can be pancreatic cancer. Inparticular embodiments, the cancer can be synovial sarcoma.

Combinations and Combination Therapy

The compounds of the present teachings can be used, alone or incombination with other pharmaceutically active compounds, to treatconditions such as those previously described hereinabove. Thecompound(s) of the present teachings and other pharmaceutically activecompound(s) can be administered simultaneously (either in the samedosage form or in separate dosage forms) or sequentially. Accordingly,in one embodiment, the present teachings comprises methods for treatinga condition by administering to the subject a therapeutically-effectiveamount of one or more compounds of the present teachings and one or moreadditional pharmaceutically active compounds.

In another embodiment, there is provided a pharmaceutical compositioncomprising one or more compounds of the present teachings, one or moreadditional pharmaceutically active compounds, and a pharmaceuticallyacceptable carrier.

In another embodiment, tire one or more additional pharmaceuticallyactive compounds is selected from the group consisting of anti-cancerdrugs, anti-proliferative drugs, and anti-inflammatory drugs.

Sigma-2 receptor binding compositions described herein are alsooptionally used in combination with other therapeutic reagents that arcselected for their therapeutic value for the condition to be treated. Ingeneral, the compounds described herein and, in embodiments wherecombination therapy is employed, other agents do not have to beadministered in the same pharmaceutical composition and, because ofdifferent physical and chemical characteristics, are optionallyadministered by different routes. The initial administration isgenerally made according to established protocols and then, based uponthe observed effects, the dosage, modes of administration and times ofadministration subsequently modified. In certain instances, it isappropriate to administer a sigma-2 receptor binding compound, asdescribed herein, in combination with another therapeutic agent. By wayof example only, the therapeutic effectiveness of a sigma-2 receptorbinding compound is enhanced by administration of another therapeuticagent (which also includes a therapeutic regimen) that also hastherapeutic benefit. Regardless of the disease, disorder or conditionbeing treated, the overall benefit experienced by the patient is eithersimply additive of the two therapeutic agents or the patient experiencesan enhanced (i.e., synergistic) benefit. Alternatively, if a compounddisclosed herein has a side effect, it can be appropriate to administeran agent, to reduce the side effect: or the therapeutic effectiveness ofa compound described herein can be enhanced by administration of anadjuvant.

Therapeutically effective dosages vary when the drugs are used intreatment combinations. Methods for experimentally determiningtherapeutically effective dosages of drugs and other agents for use incombination treatment regimens are documented methodologies. Combinationtreatment further includes periodic treatments that start and stop atvarious times to assist with the clinical management of the patient. Inany case, the multiple therapeutic agents (one of which is a sigma-2receptor binding compound as described herein) can be administered inany order, or simultaneously. If simultaneously, the multipletherapeutic agents are optionally provided in a single, unified form, orin multiple forms (by way of example only, either as a single pill or astwo separate pills).

In some embodiments, one of the therapeutic agents is given in multipledoses, or both are given as multiple doses. If not simultaneous, thetiming between the multiple doses optionally varies from more than zeroweeks to less than twelve weeks.

In addition, the combination methods, compositions and formulations arenot to be limited to the use of only two agents, the use of multipletherapeutic combinations are also envisioned. It is understood that thedosage regimen to treat, prevent, or ameliorate the condition(s) forwhich relief is sought, is optionally modified in accordance with avariety of factors. These factors include the disorder from which thesubject suffers, as well as the age, weight, sex, diet, and medicalcondition of the subject. Thus, the dosage regimen actually employedvaries widely, in some embodiments, and therefore deviates from thedosage regimens set forth herein.

The pharmaceutical agents which make up the combination therapydisclosed herein are optionally a combined dosage form or in separatedosage forms intended for substantially simultaneous administration. Thepharmaceutical agents that make up the combination therapy areoptionally also administered sequentially, with either agent beingadministered by a regimen calling for two-step administration. Thetwo-step administration regimen optionally calls for sequentialadministration of the active agents or spaced-apart administration ofthe separate active agents. The time between the multiple administrationsteps ranges from a few minutes to several hours, depending upon theproperties of each pharmaceutical agent, such as potency, solubility,bioavailability, plasma half-life and kinetic profile of thepharmaceutical agent.

In another embodiment a sigma-2 receptor binding compound inhibitor isoptionally used in combination with procedures that provide additionalbenefit to the patient. A sigma-2 receptor binding compound inhibitorand any additional therapies are optionally administered before, duringor after the occurrence of a disease or condition, and the timing ofadministering the composition containing a sigma-2 receptor bindingcompound varies in some embodiments. Thus, for example, a sigma-2receptor binding compound is used as a prophylactic and is administeredcontinuously to subjects with a propensity to develop conditions ordiseases in order to prevent the occurrence of the disease or condition.A sigma-2 receptor binding compound and compositions are optionallyadministered to a subject during or as soon as possible after the onsetof the symptoms. While embodiments of the present teachings have beenshown and described herein, it will be obvious to those skilled in theart that such embodiments are provided by way of example only. Numerousvariations, changes, and substitutions can occur to those skilled in theart without departing from the teachings. In some embodiments of thepresent teachings, various alternatives to the embodiments describedherein can be employed in practicing the present teachings.

A sigma-2 receptor binding compound can be used in combination withanti-cancer drugs, including but not limited to the following classes:alkylating agents, angiopoietin 1 and/or 2 inhibitors, anthracyclines,antimetabolite agents, aurora kinase inhibitors. B-raf inhibitors, BTKinhibitors, c-met inhibitors, CDK 4 and/or 6 inhibitors. CDK4 and/orCDK6 inhibitors, cFMS inhibitors, crosslinking agents. DMA replicationinhibitors, endothelial growth factor (EGF) inhibitors, hepatocytegrowth factor/scatter factor (HGF/SF) inhibitors, HER2 and HER3inhibitors insulin-like growth factor 1 receptor (IGFR-1) inhibitors,intercalators, MEK inhibitors, microtubule disrupters, mTOR inhibitors,pan-ErbB tyrosine kinase inhibitors, PARP inhibitors, P13K inhibitors,PKB inhibitors, PKB inhibitors, polo-like kinase inhibitors,radiomimetic agents, radiosensitizers, recombinant human apo2 ligands,strand break agents, topoisomerase II inhibitors, tumor necrosisfactor-related apoptosis-inducing ligand (TRAIL) agonist, and vascularendothelial growth factor (VEGF) inhibitors.

The compounds disclosed herein, including compounds of Formula I, arealso useful as chemo- and radio-sensitizers for cancer treatment. Theyare useful for the treatment of mammals who have previously undergone orare presently undergoing or will be undergoing treatment for cancer.Such other treatments include chemotherapy, radiation therapy, surgeryor immunotherapy, such as cancer vaccines.

The instant compounds are particularly useful in combination withtherapeutic, anti-cancer and/or radiotherapeutic agents. Thus, thepresent disclosure provides a combination of the presently compounds ofFormula I with therapeutic, anti-cancer and/or radiotherapeutic agentsfor simultaneous, separate or sequential administration. The compoundsof this disclosure and the other anticancer agent can act additively orsynergistically. A synergistic combination of the present compounds andanother anticancer agent might allow the use of lower dosages of one orboth of these agents and/or less frequent dosages of one or both of theinstant compounds and other anticancer agents and/or to administer theagents less frequently can reduce any toxicity associated with theadministration of the agents to a subject without reducing the efficacyof the agents in the treatment of cancer. In addition, a synergisticeffect might result in the improved efficacy of these agents in thetreatment of cancer and/or the reduction of any adverse or unwanted sideeffects associated with the use of either agent alone.

The therapeutic agent, anti-cancer agent and/or radiation therapy can beadministered according to therapeutic protocols well known in the art.It will be apparent to those skilled in the art that the administrationof the therapeutic agent, anti-cancer agent and/or radiation therapy canbe varied depending on the disease being treated and the known effectsof the anti-cancer agent and/or radiation therapy on that disease. Also,in accordance with the knowledge of the skilled clinician, thetherapeutic protocols (e.g., dosage amounts and times of administration)can be varied in view of the observed effects of the administeredtherapeutic agents (i.e., anti-neoplastic agent or radiation) on thepatient, and in view of the observed responses of the disease to theadministered therapeutic agents, and observed adverse effects.

Dosage ranges for x-rays range from daily doses of 50 to 200 roentgensfor prolonged periods of time (3 to 4 weeks), to single doses of 2000 to6000 roentgens. Dosage ranges for radioisotopes vary widely, and dependon the half-life of the isotope, the strength and type of radiationemitted, and the uptake by the neoplastic cells.

Any suitable means for delivering radiation to a tissue can be employedin the present disclosure. Common means of delivering radiation to atissue is by an ionizing radiation source external to the body beingtreated. Alternative methods for delivering radiation to a tissueinclude, for example, first delivering in vivo a radiolabeled antibodythat immunoreacts with an antigen of the tumor, followed by deliveringin vivo an effective amount of the radio labeled antibody to the tumor.In addition, radioisotopes can be used to deliver ionizing radiation toa tissue or cell. Additionally, the radiation can be delivered by meansof a radiomimetic agent. As used herein a “radiomimetic agent” is achemotherapeutic agent, for example melphalan, that causes the same typeof cellular damage as radiation therapy, but without the application ofradiation.

For use in cancer and neoplastic diseases a sigma-2 receptor bindingcompound can be optimally used together with one or more of thefollowing non-limiting examples of anti-cancer agents: (1) alkylatingagents, including but not limited to cisplatin (PLATIN), carboplatin(PARAPLATIN), oxaliplatin (ELOXATIN), streptozocin (ZANOSAR), busulfan(MYLKRAN) and cyclophosphamide (ENDOXAN); (2) anti-metabolites,including but not limited to mercaptopurine (PURINETHOL), thioguanine,pentostatin (NIPENT), cytosine arabinoside (ARA-C), gemcitabine(GEMZAR), fluorouracil (CARAC), leucovorin (FUSILEV) and methotrexate(RHEUMATREX): (3) plant alkaloids and terpenoids, including but notlimited to vincristine (ONCOVIN), vinblastine and paclitaxel (TAXOL);(4) topoisomerase inhibitors, including but not limited to irinotecan(CAMPTOSAR), topotecan (HYCAMTIN) and etoposide (EPOSIN); (5) cytotoxicantibiotics, including but not limited to actinomycin D (COS MEGEN),doxorubicin (ADRIA MYCIN), bleomycin (B LENOXANE) and mitomycin(MITOSOL); (6) angiogenesis inhibitors, including but not limited tosunitinib (SUTENT) and bevacizumab (AVASTIN); and (7) tyrosine kinaseinhibitors, including but not limited to imatinib (GLEEVEC), erlotinib(TARCEVA), lapatinib (TYKERB) and axitinib (INLYTA).

The additional therapeutic agent can be chosen from 5-fluorouracil,adriamycin, afatinib, alemtuzumab, altretamine, aminoglutethimide,aminolaevulinic acid, amacrine, anastrozole, aprepitant, asparaginase,axitinib, azacytidine, beg, bortezomib, bevacizumab, bexarotene,bicalutamide, bleomycin, bortezomib, bosutinib, buserelin, busulfan,camptothecin, capecitabine, carboplatin, carbozantimib, carfilzomib,carmustine, ceritinib, cetuximab, chlorambucil, chloroquine, cisplatin,cladisat, aq. NaCl solution, cladribine, clodronate, clofarabine,cobimetinib, colchicine, crizotinib, cyclophosphamide, cyclophosphamine,cyproterone, cytarabine, dacarbazine, dactinomycin, dasatinib,daunorubicin, debrafinib, decarazine, decitabine, demethoxyviridin,dasatinib, dexrazoxane, dichloroacetate, dienestrol, diethylstilbestrol,docetaxel, doxorubicin, enzalutamide, epirubicin, erlotinib, estradiol,estramustine, etoposide, everolimus, exemestane, filgrastim,fludarabine, fludrocortisone, fluorouracil, fluoxymesterone, flutamide,fulvestrant, gefitinib, gemcitabine, gemtuzumab, genistein, goserelin,hydroxyurea, ibrutinib, idarubicin, idelalisib, ifosfamide, imatinib,imiquimod, interferon, irinotecan, irinotecan, ixabepilone, lapatinib,lenalidomide, letrozole, leucovorin, leuprolide, levamisole, lomustine,lonidamine, mechlorethamine, medroxyprogesterone, megestrol, melphalan,mercaptopurine, mesna, metformin, methotrexate, mithram, mitomycin,mitomycin, mitotane, mitoxane, mitoxantrone, nelarabine, neratinib,nilotinib, nilutamide, nocodazole, octreotide, olaparib, oxaliplatin,paclitaxel, pamidronate, pazopanib, pegaspargase, pemetrexed,pemostatin, perifosine, plicamycin, pomalidomide, ponatinib, porfimer,procarbazine, raloxifene, raltitrcxed, regorafinib, rituximab,sorafenib, streptozocin, sunitinib, suramin, tamoxifen, temozolomide,temsirolimus, teniposide, testosterone, thalidomide, thioguanine,thiotepa, titanocene dichloride, topotecan, trametinib, trastuzumab,tretinoin, veliparib, vinblastine, vincristine, vindesine, vinorelbine,volasertib, vorinostat, and zoledronic acid

Where a subject is suffering from or at risk of suffering from aninflammatory condition, a sigma-2 receptor binding compound describedherein is optionally used together with one or more agents or methodsfor treating an inflammatory condition in any combination. Therapeuticagents/treatments for treating an autoimmune and/or inflammatorycondition include, but are not limited to any of the following examples:(1) corticosteroids, including but not limited to cortisone,dexamethasone, and methylprednisolone; (2) nonsteroidalanti-inflammatory drugs (NSAIDs), including but not limited toibuprofen, naproxen, acetaminophen, aspirin, fenoprofen (NALFON),flurbiprofen (ANSAID), ketoprofen, oxaprozin (DAYPRO), diclofenac sodium(VOLTAREN), diclofenac potassium (CATAFLAM), etodolac (LODINE),indomethacin (INDOCIN), ketorolac (TORADOL), sulindac (CLINORIL),tolmetin (TOLECTIN), meclofenamate (MECLOMEN), mefenamic acid (PONSTEL),nabumetone (RELAFEN) and piroxicam (FELDENE); (3) immunosuppressants,including but not limited to methotrexate (RHEUMATREX), leflunomide(ARAVA), azathioprine (IMURAN), cyclosporine (NEORAL. SANDIMMUNE),tacrolimus and cyclophosphamide (CYTOXAN): (4) CD20 blockers, includingbut not limited to rituximab (RITUXAN); (5) Tumor Necrosis Factor (TNF)blockers, including but not limited to etanercept (ENBREL), infliximab(REMICADE) and adalimumab (HUMIRA); (6) interleukin-1 receptorantagonists, including but not limited to anakinra (KINERET); (7)interleukin-6 inhibitors, including but not limited to tocilizumab(ACTEMRA); (8) interleukin-17 inhibitors, including but not limited toAIN457; (9) Janus kinase inhibitors, including but not limited totasocitinib; and (10) syk inhibitors, including but not limited tofostamatinib.

Compound Synthesis

Compounds of the present teachings can be prepared using methodsillustrated in general synthetic schemes and experimental proceduresdetailed below. General synthetic schemes and experimental proceduresare presented for purposes of illustration and are not intended to belimiting. Starting materials used to prepare compounds of the presentteachings are commercially available or can be prepared using routinemethods known in the art.

LIST OF ABBREVIATIONS

aq.=aqueous: CDCl₃-deuterated chloroform; DMSO-d₆=deuterated dimethylsulfoxide; DMSO=dimethyl sulfoxide; h=hour, THF=tetrahydrofuran.

Some embodiments of the present teachings include methods of synthesisof compounds of Formula IV:

(IV). In various configurations, these methods comprise reacting acompound of structural Formula V:

with a compound of structural formula VI:

wherein n is an integer from 1 to 5, and R² is hydrogen or methyl.

In some configurations, n can be 1; and R² can be hydrogen.

In some configurations, n can be 5: and R² can be hydrogen.

In some configurations, the reaction can take place in the presence of areducing agent, such as, without limitation, aluminum hydride,borane-tetrahydrofuran, catecholborane, diisobutylaluminum hydride,disiamylborane, hydrazine, lithium aluminum hydride, lithiumborohydride, lithium tri-t-butoxyaluminum hydride, lithiumtriethylborohydride, potassium tri-s-butylborohydride, sodiumborohydride, sodium cyanoborohydride, sodium triacetoxyborohydride, ormixtures thereof.

General Methods for Preparing Compounds

The following schemes can be used to practice synthetic methods of thepresent teachings. Additional structural groups, including but notlimited to those defined elsewhere in the specification and not shown inthe compounds described in the schemes can be incorporated to givevarious compounds disclosed herein, or intermediate compounds which can,after further manipulations using techniques known to those skilled inthe art, be converted to compounds of the present teachings.

Example 13-(o-Ethoxyphenyl)-2-{[4-(2-{p-[(6-{3-(2-methoxytoluidinocarbonyloxy)-9-azabicyclo[3.3.1]non-9-yl}hexylamino)methyl]phenoxy}acetyl)-1-piperazinyl]methyl}-3H-quinazolin-4-one)(SW V-49s)

Intermediate 1A. N-9-Benzyl-9-azabicyclo[3.3.1]nonan-3α-ol (2)

A mixture of LiAlH(o-tert-Bu)₃ (20.0 g, 78.5 mmol) in anhydrous THF (30mL) was cooled in an ice-bath. A solution of compound9-azabicyclo[3.3.1]nonan-3-one (1) (5.0 g, 21.8 mmol) in anhydrous THF(45 mL) was added drop wise. The mixture was stirred at room temperatureovernight. The reaction was quenched with saturated aqueous NH₄Cl. Thesolid was filtered off and washed with THF. The combined organic layerswere dried over Na₂SO₄, filtered and evaporated to dryness to giveN-9-Benzyl-9-azabicyclo[3.3.1]nonan-3α-ol as a light yellow oil (4.8 g,95% yield). ¹H NMR (CDCl₃) ε 7.20-7.35 (m, 5H), 4.22-4.32 (m, 1H), 3.79(s, 2H), 3.02-3.06 (m, 2H), 2.33-2.43 (m, 2H), 2.12-2.26 (m, 1H),1.85-1.99 (m, 3H), 1.30-1.54 (m, 3H), 1.08-1.13 (m, 2H).

Intermediate 1B.N-(9-Benzyl-9-azabicyclo[3.3.1]nonan-3α-yl)-N′-(2-methoxy-5-methyl-phenyl)carbamate(3)

A mixture of Intermediate 1A (4.8 g, 20.6 mmol),2-methoxy-5-methylphenyl isocyanate (3.8 g, 23.5 mmol), dibutyltindiacetate (a few drops) in CH₂Cl₂ (45 mL) was stirred at roomtemperature overnight. The reaction mixture was washed with water,saturated aqueous NaHCO₃and brine, and then dried over Na₂SO₄. Thesolvent was removed under reduced pressure and the residue was purifiedby silica gel column chromatography (hexane/ethyl acetate/triethylamine,80:20:1) to obtainN-(9-Benzyl-9-azabicyclo[3.3.1]-nonan-3α-yl)-N′-(2-methoxy-5-methyl-phenyl)carbamateas a white solid (6.8 g, 83% yield). ¹H NMR (CDCl₃) δ 7.97 (s, 1H),7.16-7.37 (m, 6H), 6.73-6.80 (m, 2H), 5.22-5.30 (m, 1H), 3.84 (s, 3H),3.81 (s, 2H), 3.04-3.07 (m, 2H), 2.42-2.52 (m, 2H), 2.30 (s, 3H),1.91-2.20 (m, 3H), 1.48-1.56 (m, 3H), 1.15-1.18 (m, 2H).

Intermediate 1C.O-(9-Azabicyclo[3.3.1]nonan-3α-yl)-N-(2-methoxy-5-methylphenyl)-car-bamate(4)

To a solution of Intermediate 1B (6.8 g, 17.3 mmol) in methanol/ethylacetate (1:1, 160 mL) was added 20% w/w Palladium hydroxide/carbon (1.35g) and ammonium formate (5.4 g, 86.5 mmol). The mixture was refluxed for6 h, cooled, filtered through a pad of celite and evaporated. Theresulting residue was dissolved in ethyl acetate, washed with saturatedaqueous NaHCO₃, water and brine, and then dries over Na₂SO₄. The solventwas removed to give the deprotected bicyclic amineO-(9-Azabicyclo[3.3.1]nonan-3α-yl)-N-(2-methoxy-5-methylphenyl)-carbamate4 as a light brown oil (quantitative). ¹H NMR (CDCl₃) δ 7.94 (s, 1H),7.16 (s, 1H), 6.73-6.80 (m, 2H), 4.96-5.04 (m, 1H), 3.84 (s, 3H),3.33-3.36 (m, 2H), 2.33-2.41 (m, 2H), 2.30 (s, 3H), 2.06-2.14 (m, 1H),1.45-1.75 (m, 8H).

Intermediate 1D.N-(9-(6-aminohexyl)-9-azabicyclo[3.3.1]nonan-3α-yl)-N′-(2-methoxy-5-methyl-phenyl)carbamate (6a)

A mixture of secondary amine from Intermediate 1C (3.3 g, 11.0 mmol),N-(6-bromohexyl)phthalimide (3.5 g, 11.3 mmol). KI (2.0 g, 12.4 mmol)and K₂CO₃ (7.8 g, 56.5 mmol) in acetonitrile (90 mL) was stirred atreflux overnight. After filtration, volatile components were evaporatedin vacuo. The resulting residue was purified by silica gel columnchromatography (5% methanol in dichloromethane) to give the desiredintermediate phthalimido-protected amine 5a (5.8 g, 96% yield) as alight brown oil.

Compound 5a (2.8 g, 5.3 mmol) was refluxed with hydrazine hydrate (540mg, 10.7 mmol) in ethanol (100 mL) for 5 h. The solvent was evaporatedand 10% aqueous solution of NaOH (20 mL) was added. The mixture wasextracted with CH₂C₂, dried over Na₂SO₄, and evaporated to give thedesired primary amineN-(9-(6-aminohexyl)-9-azabicyclo[3.3.1]nonan-3α-yl)-N′-(2-methoxy-5-methyl-phenyl)carbamate 6a (1.9 g, 88% yield) as a light yellow oil. ¹H NMR (CDCl₃) δ7.96 (s, 1H), 7.14 (s, 1H), 6.72-6.80 (m, 2H), 5.10-5.18 (m, 1H), 3.84(s, 3H), 3.05-3.07 (m, 2H), 2.66-2.71 (m, 2H), 2.55-2.59 (m, 2H),2.39-2.49 (m, 2H), 2.29 (s, 3H), 2.10-2.20 (m, 1H), 1.81-1.94 (m, 2H),1.18-1.54 (m, 15H).

Intermediate 1E. Ethyl 2-(4-formylphenoxy)acetate(7)

Ethyl 2-bromoacetate (3.7 g, 22.0 mmol)) and potassium carbonate (8.3 g,60.0 mmol) were added into the solution of 4-hydroxybenzaldehyde (2.4 g,20.0 mmol) in acetonitrile (60 mL). The reaction mixture was refluxedfor 24 h. After cooling, the reaction mixture was filtered, andevaporated to give ethyl 2-(4-formylphenoxy)acetate as a light yellowliquid (quantitative). ¹H NMR (CDCl₃) δ 9.90 (s, 1H), 7.85 (d, J=8.4 Hz,2H), 7.01 (d, J=8.4 Hz, 2H), 4.71 (s, 2H), 4.28 (q, J=7.1 Hz, 2H), 1.31(t, J=7-7.1 Hz, 3H).

Intermediate 1F. 2-(4-Formylphenoxy)acetic acid (8)

Hydrolysis of Intermediate 1E with sodium hydroxide (2.2 eq) inmethanol/water (2:1, 90 mL) for 24 h, followed by acidifying with 10%HCl solution gave 2-(4-Formylphenoxy)acetic acid as an off-white solid(3.1 g, 87% yield). ¹H NMR (DMSO-d₆) δ 13.18 (br s, 1H), 9.91 (s, 1H),7.89 (d, 8.6 Hz, 2H), 7.13 (d, J=8.6 Hz, 2H), 4.86 (s, 2H).

Intermediate 1G. 2-(2-Chloroacetamido)benzoic acid (9)

Triethylamine (4.06 g, 40.1 mmol) was added in the solution of2-aminobenzoic acid (5.0 g, 36.5 mmol) in dichloromethane (90 mL) andthe mixture was cooled in an ice-water bath, A solution of chloroacetylchloride (4.5 g, 40.1 mmol) in dichloromethane (40 mL) was added dropwise and the mixture was allowed to stir at ambient temperatureovernight. The solids were filtered and washed with cold water followedby 5% diethyl ether in hexane and were air dried to afford2-(2-chloroacetamido)benzoic acid as a white solid (7.4 g, 95% yield).¹H NMR (DMSO-d₆) 11.82 (s, 1H), 8.52 (d, J=7.5 Hz, 1H), 8.01 (d, J=7.5Hz, 1H), 7.63 (t, J=7.5 Hz, 1H), 7.21 (t, J=7.5 Hz, 1H), 4.45 (s, 2H).

Intermediate 1H.2-(Chloromethyl)-3-(2-ethoxyphenyl)quinazolin-4(3H)-one(10)

Phosphoryl chloride (6.5 g, 42.4 mmol) was added drop wise to a mixtureof Intermediate 1G (3.1 g, 14.5 mmol) and 2-ethoxyaniline (2.0 g, 14.5mmol) in acetonitrile (50 mL). The mixture was heated at refluxovernight. The reaction mixture was cooled to room temperature, pouredinto a slurry of ice/saturated solution of Na₂CO₃. The resulting solidwas filtered, washed with water and air dried to give2-(chloromethyl)-3-(2-ethoxyphenyl)quinazolin-4(3H)-one (10) as a brownsolid (2.5 g, 53% yield). ¹H NMR (CDCl₃) δ 8.31 (d, J=7.8 Hz, 1H),7.78-7.82 (m, 2H), 7.47-7.55 (m, 2H), 7.35 (d, J=7.4 Hz, 1H), 7.06-7.14(m, 2H), 4.35 (d, J=11.9 Hz, 1H), 4.17 (d, J=11.9 Hz, 1H), 4.06 (q,J=—6.9 Hz, 2H), 1.23 (t, J=6.9 Hz, 3H).

Intermediate11.3-(2-Ethoxyphenyl)-2-(piperazin-1-yl-methyl)quinazolin-4(3M)-one (11)

Piperazine (2.7 g, 32.0 mmol) was added to a mixture of Intermediate 1H(2.5 g, 8.0 mmol), K₂CO₃(4.4 g, 32.0 mmol) and KI (1.7 g, 10.4 mmol) inacetonitrile (75 mL). The reaction mixture was heated at 85-90° C.,overnight. After cooling, it was filtered and the solid was washed withacetonitrile. The combined organic layers were evaporated. The resultingresidue was dispersed in water and extracted with ethyl acetate. Theorganic layers were washed with brine, dried over Na₂SO₄, filtered andevaporated. The crude residue was purified by column chromatography (10%methanol, 0.5% NH₄OH in dichloromethane) to give3-(2-ethoxyphenyl)-2-(piperazin-1-yl-methyl)quinazolin-4(3H)-one as ayellow oil (2.3 g, 79% yield). ¹H NMR (CDCl₃) δ 8.30 (d, J=7.8 Hz, 1H),7.75-7.77 (m, 2H), 7.39-7.50 (m, 2H), 7.28 (d, J=7.0 Hz, 1H), 7.01-7.08(m, 2H), 4.05 (q, J=6.8 Hz, 2H), 3.18-3.28 (m, 2H), 2.73 (s, 4H),2.33-2.37 (m, 2H), 2.17-2.20 (m, 2H), 1.22 (t, J=6.8 Hz, 3H).

Intermediate 1J.4-(2-(4-((3-(2-Ethoxyphenyl)-4-oxo-3,4-dihydroquinazolin-2-yl)methyl)-piperazin-1-yl)-2-oxoethoxy)benzaldehyde(12)

A cooled mixture of acid 8 (360 mg, 2.0 mmol), N-hydroxysuccinimide (280mg, 2.4 mmol) in acetonitrile (12 mL) was added a solution of DCC (500mg, 2.4 mmol) in acetonitrile (4 mL). After stirring at room temperaturefor 45 min, a solution of Intermediate 11 (800 mg, 2.2 mmol) inacetonitrile (10 mL) was added, then continued stirring overnight. Thesolid was filtered off and the filtrate was evaporated. The resultingresidue was purified by column chromatography (8% methanol indichloromethane) to give4-(2-(4-((3-(2-Ethoxyphenyl)-4-oxo-3,4-dihydroquinazolin-2-yl)methyl)-piperazin-1-yl)-2-oxo-ethoxy)benzaldehydeas an off-white solid (847 mg, 80% yield). ¹H NMR (CDCl₃) δ 9.89 (s,1H), 8.27-8.31 (m, 1H), 7.72-7.84 (m, 4H), 7.42-7.52 (m, 2H), 7.25 (d,J=6.3 Hz, 1H), 7.02-7.09 (m, 4H) 4.74 (s, 2H), 4.05 (q, J=7.0 Hz, 2H),3.42-3.49 (m, 4H), 3.22-3.31 (m, 2H), 2.18-2.46 (m, 4H), 1.22 (t, J=7.0Hz, 3H).

9-(6-(((4-(2-(4-((3-(2-Ethoxyphenyl-4-oxo-3,4-dihydroquinazolin-2-yl)methyl)-piperazin-1-yl)-2-oxoethoxy)benzyl)amino)hexyl)-N-(9-azabicyclo[3.3.1]nonan-3α-yl)-N-(2-methoxy-5-methylphenyl)carbamateoxalate salt (SW V-49s)

A solution of amine Intermediate 1D (6a) (386 mg, 0.95 mmol) indichloromethane (4 mL) was added into a solution of Intermediate 1J (12)(480 mg, 0.91 mmol) in dichloromethane (4 mL). The mixture was stirredfor 4 h then the solvent was evaporated. The residue was dissolved inethanol (5 mL), then NaBH₄ (100 mg, 2.6 mmol) was added. The mixture wasstirred few 6 h, then quenched with 10% HCl solution. After the solventwas evaporated, it was basified with 10% NaOH solution, extracted withdichloromethane and evaporated. The resulting residue was purified bycolumn chromatography (10% methanol, 0.5% NH₄OH in dichloromethane) togive the product as free amine (460 mg, 55% yield). ¹H NMR (CDCl₃) δ8.30 (d, J=7.8 Hz, 1H), 7.94 (s, 1H), 7.74-7.78 (m, 2H), 7.41-7.51 (m,2H), 7.23-7.26 (m, 3H), 7.14 (s, 1H), 7.03-7.09 (m, 2H), 6.86 (d, J=8.6Hz, 2H), 6.73-6.79 (m, 2H), 5.10-5.16 (m, 1H), 4.62 (s, 2H), 4.04 (q,J=6.9 Hz, 2H), 3.85 (s, 3H), 3.74 (s, 2H), 3.43-3.48 (m, 4H), 3.22-3.30(m, 2H), 3.12 (br s; 2H), 2.60-2.64 (m, 4H), 2.37-2.50 (m, 4H), 2.29 (s,3H), 2.19-2.25 (m, 3H), 1.90-1.96 (m, 2H), 1.48-1.58 (m, 7H), 1.28-1.34(m, 6H), 1.22 (t, J-=6.9 Hz, 3H). The oxalate salt was prepared using 1equivalent of oxalic acid in ethanol to give SW V-49s as an off-whitesolid (470 mg, 93% yield), mp 164-165° C. Anal. (C₅₅H₆₉N₇O₁₁.2H₂O):Calculated, %: C, 63.51: H, 7.07; N, 9.43, Found, %: C, 63.54, H, 7.06,N, 9.76.

Example 23-(o-Ethoxyphenyl)-2-{[4-(2-{p-[(10-{3-(2-methoxytoluidinocarbonyloxy)-9-azabicyclo[3.3.1]non-9-yl}decylamino)methyl)phenoxy}acetyl)-1-piperazinyl]methyl}-3H-quinazolin-4-one)oxalate salt. (SW V-50s)

Intermediate 2A.N-(9-(10-aminodecyl)-9-azabicyclo[3.3.1]nonan-3α-yl-(2-methoxy-5-methylphenyl)carbamate (6b)

A mixture of secondary amine from Intermediate 1C (3.6 g, 11.8 mmol),N-(10-bromodecyl)phthalimide (4.4 g, 12.0 mmol). KI (2.0 g, 12.4 mmol)and K₂CO₃ (8.2 g, 59.4 mmol) in acetonitrile (90 mL) was stirred atreflux overnight. After filtration, volatile components were evaporatedin vacuo. The resulting residue was purified by silica gel columnchromatography (5% methanol in dichloromethane) to give the desiredphthalimido-protected intermediate 5b (6.0 g, 86% yield) as a lightbrown oil.

Compound 5b (2.9 g, 4.9 mmol) was refluxed with hydrazine hydrate (700mg, 13.9 mmol) in ethanol (100 mL) for 5 h. The solvent was evaporatedand 10% aqueous solution of NaOH (20 mL) was added. The mixture wasextracted with CH₂Cl₂, dried over Na₂SO₄, and evaporated to give theprimary amineN-(9-(10-aminodecyl)-9-azabicyclo[3.3.1]nonan-3α-yl)-N′-(2-methoxy-5-methylphenyl)carbamate 6b (2.2 g, 95% yield) as a light yellow oil. ¹H NMR (CDCl₃) δ7.96 (s, 1H), 7.14 (s, 1H), 6.72-6.80 (m, 2H), 5.10-5.18 (m, 1H), 3.84(s, 3H), 3.04-3.07 (m, 2H), 2.65-2.70 (m, 2H), 2.53-2.58 (m, 2H),2.39-2.49 (m, 2H), 2.29 (s, 3H), 2.08-2.20 (m, 1H), 1.83-1.94 (m, 2H),1.18-1.54 (m, 23H).

A solution of amine Intermediate 2 A (343 mg, 0.74 mmol) indichloromethane (4 mL) was added into a solution of 12 (380 mg, 0.72mmol) in dichloromethane (4 mL). The mixture was stirred for 4 h thenthe solvent was evaporated. The residue was dissolved in ethanol (5 mL),then NaBH₄ (100 mg, 2.6 mmol) was added. The mixture was stirred for 6h, then quenched with 10% HCl solution. After the solvent wasevaporated, it was basified with 10% NaOH solution, extracted withdichloromethane and evaporated. The resulting residue was purified bycolumn chromatography (10% methanol, 0.5% NH₄OH in dichloromethane) togive the product as free amine (295 mg, 42% yield). ¹H NMR (CDCl₃) δ8.29 7.8 Hz, 1H), 7.95 (s, 1H), 7.73-7.78 (m, 2H), 7.41-7.51 (m, 2H),7.21-7.24 (m, 3H), 7.14 (s, 1H), 7.02-7.08 (m, 2H), 6.86 (d, J=8.2 Hz,2H), 6.73-6.79 On, 2H), 5.10-5.16 (m, 1H), 4.61 (s, 2H), 4.04 (q, J=6.9Hz, 2H), 3.84 (s, 3H), 3.72 (s, 2H), 3.44-3.48 (m, 4H), 3.21-3.31 (m,2H), 3.09 (br s, 2H), 2.59-2.62 (m, 4H), 2.36-2.50 (m, 4H), 2.29 (s,3H), 2.17-2.24 (m, 3H), 1.88-1.94 (m, 2H), 1.40-1.57 (m, 7H), 1.24-1.30(m, 6H), 1.21 (t, J=6.9 Hz, 3H). The oxalate salt was prepared using 1equivalent of oxalic acid in ethanol to give SW V-50s as a light brownsolid (308 mg, 95% yield), mp 183-184° C. Anal. (C₅₉H₇₇N₇O₁₁.2H₂O):Calculated. %: C, 64.64; H, 7.45; N, 8.94, Found, %: C, 64.84. H, 7.41,N, 8.62.

TABLE 1 Additional Examples of dual-domain sigma-2 receptor ligand drugconjugate compounds, including sigma-2 receptor ligand erastin conjugatecompounds and sigma-2 receptor ligand erastin-analog conjugatecompounds. Ex. No. Structure  3

 4

 5

 6

 7

 8

 9

10

11

12

13

14

15

16

17

18

18

20

21

22

23

24

Biological Activity Assays

The following are assays that can be used to evaluate the biologicalefficacy of compounds of Formula (1).

The methods and compositions described herein utilize laboratorytechniques well known to skilled artisans, and can be found inlaboratory manuals such as Sambrook. J., et al., Molecular Cloning: ALaboratory Manual, 3rd ed. Cold Spring Harbor Laboratory Press. ColdSpring Harbor, N.Y., 2001; Spector, D. L. et al., Cells: A LaboratoryManual, Cold Spring Harbor Laboratory Press. Cold Spring Harbor, N.Y.,1998; Nagy, A., Manipulating the Mouse Embryo: A laboratory Manual(Third Edition). Cold Spring Harbor, N.Y., 2003; Harlow, E., UsingAntibodies: A laboratory Manual, Cold Spring Harbor Laboratory Press.Cold Spring Harbor. N.Y., 1999; and Carruthers, W., and Coldham, I.,Modem Methods of Organic Synthesis (4th Edition), Cambridge UniversityPress, Cambridge, U.K., 2004. Methods of administration ofpharmaceuticals and dosage regimes, can be determined according tostandard principles of pharmacology well known skilled artisans, usingmethods provided by standard reference texts such as Remington: theScience and Practice of Pharmacy (Alfonso R. Gennaro ed. 19th ed. 1995);Hardman, J. G., et al., Goodman & Gilman's The Pharmacological Basis ofTherapeutics, Ninth Edition. McGraw-Hill, 1996; and Rowe, R. C., et at.,Handbook of Pharmaceutical Excipients. Fourth Edition. PharmaceuticalPress, 2003. As used in the present description and the appended claims,the singular forms “a”. “an” and “the” are intended to include theplural forms as well, unless the context indicates otherwise.

Toxicity Study Methods

Six adult female mice were submitted to necropsy. The strain designationof the mice was C57BL/6. Previous procedures included dailyintraperitoneal administration of a ferroptosis-inducing drug or vehiclecontrol over the course of 2 weeks. Mice were also previouslytransplanted subcutaneously with a KCKO xenograft (Besmer, D. M., etal., Cancer Res. 17: 4432-4442, 2011).

Cell Lines

CFPAC-1, BxPC-3, AsPC-1, PANC-1 and Mia PaCa-2 cell lines were obtainedfrom American Type Culture Collection (ATCC, Manassas, Va.). SYO-1 cellline, a synovial sarcoma cell line (Kawai, A., et al., Cancer Lett. 204:105-113.2004), was provided by Dr. Brian Van Tine (Washington UniversitySchool of Medicine. St. Louis, Mo.). KCKO cell line was isolated from ahuman MUC1 expressing pancreatic tumor of transgenic mouse (Besmer etal. Cancer Research 2011; 71: 4432-4442. Tinder et al. J Immunol 2008;181: 3116-3125). The KCKO cell line was provided by Dr. Pinku Mukherjee(University of North Carolina, Charlotte. N.C.). PANC-1 cells werecultured in Dulbecco's Modified Eagle's Medium with 4 mM L-glutamine,1.5 g/L Sodium bicarbonate, and 10% fetal bovine serum (FBS). Mia PaCa-2cell line was cultured in Dulbecco's Modified Eagle's Medium with 10%FBS and 2.5% horse serum. BxPC-3, AsPC-1 and KCKO cell lines werecultured in RPMI-1640 medium with 10% FBS. SYO-1 synovial sarcoma cellswore cultured in Dulbecco's Modified Eagle's Medium with 10% FBS.Antibiotics, penicillin (100 mg/ml) and streptomycin (100 mg/ml) wereadded to the media and cells were maintained in humidified incubator at37° C. with 5% CO₂.

Statistics

Statistical analyses and data plotting were performed using GraphPadPrism software version 6 (San Diego. Calif.). Results were expressed asmean±SEM of at least 3 biological replicates.) IC₅₀ values werecalculated by curve fitting normalized viability versus drugconcentration. One-way ANOVA was used to analyze the differences in IC₅₀values and SW V-49s inhibition with NAC and ZVAD tests. Unpaired twotailed t-test was used to evaluate the difference in CBC andbiochemistry analyses, and to confirm the difference in the subgroups ofcystine uptake, caspases, and ROS detection assays. Two-way ANOVA wasused to analyze the difference in tumor volume. Kaplan-Meier survivalanalysis was used and the difference between the groups was comparedwith a log-rank test. A p-value <0.05 was considered significant for allanalyses.

Pre-Necropsy Examination.

Mouse A (ID: 732) was a vehicle control. The hair coat was shaved overthe right flank, with partial regrowth. A very small, subcutaneousthickening was palpable in the right flank. There was no nasal or oculardischarge or diarrhea. Hydration and body fat were normal. Body weightwas 21 grams.

Mouse B (ID: 727) was treated with test drug. The hair coat was shavedover the right flank, with partial regrowth. A subcutaneous mass thatmeasured 1.5×0.6 cm was noted over the lumbar spine, with tightadherence into the underlying musculature. A 4-5 mm diameter ulcer wasnoted in the skin overlying this mass. There was no nasal or oculardischarge or diarrhea. Hydration and body fat were normal Body weightwas 21 grams.

Mouse C (ID: 735) was a vehicle control. Posterior paralysis was notedin this animal, with dragging of the rear limbs. Deep pain responsecould be elicited from the left rear leg, but not from the right rearleg. The hair coat was slightly thinned over the right flank. A firm,subcutaneous mass was noted over the lumbar spine. This was firmlyattached to underlying tissues. A 0.2-0.3 cm diameter thickening of theskin within which was a 0.1 cm diameter ulcer was noted over the rightflank. There was no nasal or ocular discharge or diarrhea. Hydration andbody fat were normal. Body weight was 20 grams.

Mouse D (ID: 737) was treated with test drug. A subcutaneous mass waspalpable over the spine. The hair coat was normal. There was no nasal orocular discharge or diarrhea. Hydration and body fat were normal. Bodyweight was 19 grams.

Mouse E (ID: 746) was a vehicle control. The hair coat was shaved overthe right flank, with partial regrowth. A 0.6 cm diameter firmsubcutaneous nodule was noted in the right flank. There was no nasal orocular discharge or diarrhea. Hydration and body fat were normal. Bodyweight was 23 grams.

Mouse F (ID: 738) was treated with test drug. The hair coat was normal.A firm subcutaneous mass was palpable overlying the lumbar spine. Thespine was easily palpable, suggesting possible muscle wasting. There wasno nasal or ocular discharge or diarrhea. Hydration and body fat werenormal. Body weight was 22 grams.

Gross Necropsy Examination.

Regarding Mouse A, the subcutaneous thickening noted on pre-necropsyexamination was a firm mass that measures 0.5×0.1 cm. A 0.4×0.1 cmthickened area was noted in the mesentery near the distal colon, andwithout being limited by theory, possibly the result of an enlargedlymph node. A 2 mm diameter reddened focus was noted in the left lung.The heart and liver were mildly pale. There were no gross lesions in theintestinal tract, musculoskeletal system, urinary system, genitalsystem, brain, thymus, spleen, adrenal, thyroid, pituitary, middle ear,or eye.

Regarding Mouse B, the subcutaneous mass was multilobulated, andmeasured 1.8×0.6 cm, with an attached 1.0 cm diameter nodule. Thesubcutaneous mass was located subcutaneously over the lumbar spine andright lateral dorsum. There was no infiltration into the spine. The massprojected ventrally impinging upon the abdomen. The liver was slightlypale. The heart was mildly pale. There were no gross lesions in therespiratory system, intestinal tract, urinary system, genital system,brain, thymus, spleen, lymph nodes, adrenal, thyroid, pituitary, middleear, or eye.

Regarding Mouse C, the subcutaneous mass measured 1.8×1.7×1.5 cm andappeared to encompass the mid-lumbar spine. The mass was pale-tan, firmand slightly nodular. The heart and liver were mildly pale. There wereno gross lesions in the respiratory system, intestinal tract, urinarysystem, genital system, brain, thymus, spleen, lymph nodes, adrenal,thyroid, pituitary, middle ear, or eye.

Regarding Mouse O, the subcutaneous mass measured 1.2×0.8 cm and layover the lumbar spine. Without being limited by theory, the dorsalspinous processes of the vertebrae can have been eroded by the tumor.The liver was mildly pale. There were no gross lesions in therespiratory system, intestinal tract, urinary system, genital system,heart, brain, thymus, spleen, lymph nodes, adrenal, thyroid, pituitary,middle ear, or eye.

Regarding Mouse E, the lungs were mottled red in color. The liver wasslightly pale. There were no gross lesions in the intestinal tract,musculoskeletal system, urinary system, genital system, heart, brain,thymus, spleen, lymph nodes, adrenal, thyroid, pituitary, middle car, oreye.

Regarding Mouse F, there were two masses noted in the skin andsubcutaneous tissues. One measured 0.5×0.5×0.3 cm and was located in theskin, and the other measured 0.5×0.5×0.2 cm and was in the subcutaneoustissues overlying the lumbar spine. There were no gross lesions in therespiratory system, intestinal tract, urinary system, genital system,heart, brain, thymus, spleen, lymph nodes, adrenal, thyroid, pituitary,middle ear, or eye.

Histopathologic Examination.

Regarding Mouse A, mild, multifocal inflammatory infiltrates were notedin the mesenteric fat that included macrophages, neutrophils,lymphocytes, and plasma cells. A reactive mesenteric lymph node wasnoted, with lymphoid hyperplasia, histiocytosis and dilatation of themedullary sinuses. In the lung there was moderate focal hemorrhage notedin one lobe, without limited by theory, likely related to CO₂euthanasia. There were no significant lesions in the brain, heart,liver, kidney, spleen, pancreas, or gastrointestinal tract.

Regarding Mouse B, mild to moderate chronic peritonitis was noted in themesentery and along the serosal surfaces of the small and largeintestinal tract and stomach, as well as around the pancreas. This wascharacterized by infiltrates of macrophages, neutrophils, lymphocytes,and plasma cells, along with areas of fibrosis. Mild mucosal hyperplasiawas noted in the ileum. A mesenteric lymph node located near thepancreas was reactive, as described previously. No lesions were notedwithin in the pancreatic parenchyma. Mild peritonitis was noted aroundthe gall bladder and along the capsular surface of the right kidney. Noother lesions were noted in the liver or kidneys. Mild capsularthickening and mesothelial hyperplasia were noted along the spleen.There was a moderate increase in extramedullary erythropoiesis in thered pulp of the spleen. There were no significant lesions in the brain,heart, or lungs.

Regarding Mouse C, examination of the lungs revealed a few small foci ofmetastasis of the primary tumor. Without being limited by theory, theprimary tumor appeared to have invaded bone, as small bone fragmentswere noted within the mass. There were no significant lesions in thebrain, heart, liver, kidney, spleen, pancreas, or gastrointestinaltract.

Regarding Mouse D, minimal to mild chronic peritonitis was noted alongthe serosal surface of the stomach, intestinal tract, and around thepancreas, with inflammatory cells and areas of fibrosis, as describedpreviously. Foci of peritonitis were also noted along the capsularsurfaces of the liver and kidneys. No other lesions were noted in thegastrointestinal tract, pancreas, liver, and kidneys. In the spleenthere was a moderate increase in extramedullary erythropoiesis. In thelung there were 2 small foci of pyogranuloma noted in one lung lobe.This was of unknown etiology; no foreign material was noted inassociation. Tumor invasion into the lumbar musculature was evident.Tumor cells also closely approached the vertebral body and surrounded asmall bone fragment, without being limited by theory, presumed torepresent a dorsal spinous process of the vertebral body. There were nosignificant lesions in the brain or heart.

Regarding Mouse E, in the mesenteric fat there were minimal to mildinfiltrates of macrophages, lymphocytes, neutrophils, and plasma cells.A mildly reactive lymph node was noted near the pancreas, as describedpreviously. There were no significant lesions noted in the brain, heart,lung, liver, kidneys, pancreas, spleen, or gastrointestinal tract.

Regarding Mouse F, mild chronic peritonitis was noted along the serosalsurfaces of the intestinal tract and in the mesentery surrounding thepancreas, as described previously. In one pancreatic lobule there wasloss of acinar cells and replacement by macrophages and fibroblasts,presumably representing an extension of the reaction noted in themesentery. Mild peritonitis was also noted on the capsule and in themesentery surrounding the kidneys. No other lesions were noted in theintestinal tract, liver, or kidneys. In the spleen there was a moderateincrease in extramedullary erythropoiesis and granulopoiesis. There wereno gross lesions in the brain, heart, lungs, or stomach.

Hematological Testing.

A difference noted between the drug-treated and control animals was thepresence of minimal to moderate chronic peritonitis along the serosalsurfaces of the intestinal tract and abdominal organs and in themesentery surrounding the pancreas, noted in the drug-treated animals.Whereas the control animals showed minimal to mild multifocalinflammatory infiltrates in the mesentery, without fibroplasia. Otherfindings included elevation of ALT and AST noted in mouse C. Withoutbeing limited by theory, the etiology of this finding was not clear.There was not histologic evidence of hepatocellular injury, as might beexpected. Without being limited by theory, other potential causesincluded hemolysis of the blood sample, or bone injury from tumorinvasion.

In mouse C there was tumor invasion into lumbar musculature, withfragmentation of bone, without being limited by theory, likely that of avertebral process, as well as pulmonary metastasis. In mouse D there wasinvasion of tumor into the lumbar musculature, and fragmentation of thedorsal spinous process.

TABLE 2 Complete Blood Count Results WBC RBC HGB PCV MCV MCH MCHCPlatelets ID (10³/μL) (10⁶/μL) (g/dl) (%) (u) (pg) (%) (10³/μL A-7327.72 9.15 12.4 49.2 53.8 13.6 25.2 651 B-735 8.10 8.89 11.2 45.6 51.312.6 24.6 745 C-746 5.54 8.89 11.5 44.7 50.3 12.9 25.7 788 D-727 4.167.66 9.8 37.7 49.2 12.8 26.0 611 E-737 5.68 8.67 10.7 39.3 45.3 12.327.2  749* F-738 3.34 8.40 10.5 42.4 50.5 12.5 24.8 885 *Mild plateletclumping was noted.

TABLE 3 Differential Results Neutrophils Lymphocytes MonocytesEosinophils Basophils Bands Nrbe ID (%) (%) (%) (%) (%) (%) (%) A732 1483 3 0 0 0 0 B-735 46 49 5 0 0 0 0 C-746 13 81 6 0 0 0 0 D727 13 87 0 00 0 0 E737 2 96 2 0 0 0 0 F-738 18 78 4 0 0 0 0

TABLE 4 Clinical Chemistry Results Total BUN Creatinine ALT AST GlucoseProtein ID (mg/dL) (mg/dL) (u/L) (u/L) (mg/dL) (g/dL) A-732 23 0.33 9587 179 6.1 B-735 17 0.26 65 73 180 5.4 C-746 28 0.28 506 715 187 5.5D-727 19 0.22 138 167 178 5.1 E-737 22 0.21 64 147 310 5.7 F-738 17 0.2463 55 180 5.1 Sigma-2 Receptor Binding Properties of Compound SW V-49s

In these experiments, competitive binding assays of SW V-49s with thefluorescently labeled sigma-2 ligand SW 120 of structure

were conducted.

AsPC-1 cells (5×10⁵/well) were seeded into a 6 well plate for 24 hoursbefore treatment. The cells were then incubated with 0, 10, 30 and 50 LMof SW V-49s for 30 minutes at 37° C. Subsequently, 10 nM offluorescently labeled sigma-2 ligand SW120 was added to the cell culturemedium containing SW V-49s. After 30 minute of incubation at 37° C.,cells were washed twice with phosphate buffered saline (PBS) andharvested with 0.05% trypsin EDTA (Life Technologies. Grand Island,N.Y.). Thereafter, the cells were centrifuged at 1000×g for 5 minutesand the pellets were washed twice with PBS. Internalization of SW 120was determined by flow cytometer (FACSCalibur™, BD Biosciences, SanJose, Calif.).

FIG. 3 shows competitive inhibition of internalization of 10 μM offluorescently labeled SW 120 (Spitzer. D., et al. Cancer Res.72:201-209.2012) vs. SW V-49s in human Pane-1 cells. These data indicateefficient blocking of SW 120 internalization with increasingconcentrations of SW V-49s. These results indicate that SW V-49sefficiently binds sigma-2 receptors.

SW V-49s Exhibits Lethality Towards Cancer Cell Lines In Vitro.

In these experiments, viability assays after 24 hours treatment of humanand murine pancreatic cancer cell lines and synovial sarcoma cell lineswith SW V-49s or its parent compounds SV119 and Erastin, both singly andin combination, were performed. The data demonstrate an IC₅₀concentration of 4.3 μM for SW V-49s against human pancreatic-cancercell line Pane-1 (ATCC® CRL-1469™) (FIG. 4A): an IC₅₀ concentration of2.3 μM for SW V-49s against for human pancreatic cancer cell line BxPC-3(ATCC® CRL-1687™) (FIG. 4B); an IC₅₀ concentration of 2.3 μM for SWV-49s against human pancreatic cancer cell line MIA PaCa-2 (ATCC®CRL-1420™) (FIG. 4C); an IC₅₀ concentration of 3 μM for SW V-49s againsthuman pancreatic cancer cell line AsPC-1 (ATCC® CRL-1682™) (FIG. 4D);and an IC₅₀ concentration of 2.2 μM for SW V-49s against a murinepancreatic cancer cell line (KCKO cells) (FIG. 4E). In comparison.Erastin alone exhibited an IC₅₀>100 μM against all human and murinepancreatic cancer cell lines tested: SV119 alone exhibited an IC₅₀>54 μMagainst all human and murine pancreatic cancer cell lines tested: andequimolar mixture of Erastin and SV119 exhibited an IC₅₀>44 μM againstall human and murine pancreatic cancer cell lines tested.

Synovial sarcoma cell lines (FIG. 4F, SYO-1, ME1 deficient) are sosensitive to SW V-49s that it was difficult to measure the minimal drugconcentration required to kill 50% of the cancer cells, but the measuredIC₅₀ was approx. 1.0 μM. In comparison, IC₅₀'s for other compoundstested against synovial sarcoma cells were as follows: Erastin, >16 μM;SV119, 6.2 μM; Erastin+SV119, 4.9 μM.

These data indicate that SW V-49s is far more lethal against cancercells, including human and murine pancreatic cancer cells and Synovialsarcoma cells, compared to its component parent compounds, eitherindividually or in combination.

Dual Functionality of SW V49s.

In these experiments, the inventors investigated whether compounds ofthe present teachings were lethal towards pancreatic cancer cells bytriggering an apoptosis cell death pathway, a ferroptosis cell deathpathway, or a combination thereof. The inventors thus performed assaysfor caspase 3/7 as an indicator of apoptotic cell death, and assays forthe generation of reactive oxygen species (ROS) as an indicator offerropoptotic cell death. As shown in FIG. 5A, 24 hr. treatment ofAsPC-1 cells with 50 μM Erastin, SV119 or an equimolar mixture ofErastin plus SV119 led to far less caspase activity compared to 4 μM SWV-49s. In FIG. 5B, results are shown for Caspase-Glo® (Promega) assaysof Aspc-1 cells treated with 8 μM SW V-49s, 8 μM Erastin, 8 μM SV119, or8 μM of an equimolar mixture of Erastin and SV 119, for 7 hrs. Cellstreated with SW V-49s had a significant increase (approximately 3 fold)in caspase 3/7 activity compared to all controls *p<0.0001.

In FIG. 6, results are shown for reactive oxygen species (ROS) assays ofAspc-1 treated with 8 μM 8 μM SW V-49s, 8 μM Erastin, 8 μM SV119, or 8μM of an equimolar mixture of Erastin and SV119, or a positive controlfor 30 min. Cells treated with SW V-49s had a significant (50%) increasein ROS caspase *p<0.0001 compared to Erastin, SV119 or a combinationthereof, consistent with ferropoptotic cell death. Parent compoundsSV119 and Erastin, applied to cells either singly or in combination, hadno effect at the same concentration.

Without being limited by theory, these data indicate that SW V-49sinduces both apoptotic and ferropoptotic cell death pathways inpancreatic cancer cells.

Administration of SW V-49s can Decrease Pancreatic Tumor Size In Vivo.

FIG. 7 illustrates changes in mean tumor volume (in mm³) followingadministration to interval of C57BL/6 mice with established, syngeneic,subcutaneous KCKO mouse pancreatic tumors over a period of 10 days forSW V-49s. Erastin, SV119, a mixture of Erastin and SV119, and a vehiclecontrol. SW V-49s treatment, but no other treatment, led to asignificant decrease in tumor volume (p<0.05). These treatments resultedin minimal off-target effects. Note in FIG. 7 the decrease in tumorvolume from approx. 40 mm³ to approx. 20 mm³ in SW V 49s-treated mice,compared to an increase from approx. 40 mm³ to approx. 140 mm³ for othertreatments.

Administration of SW V-49s can Increase Survival of Pancreatic Cancer ina Murine Model System.

In these experiments, in a survival study of the mice reported inExample 9, the group which received SW V-49s survived at 100% (FIG. 8).In all other groups, the mean survival clustered at around 18 days.

SW V-49s Induces Cell Death in Pancreatic Cancer.

Cytotoxicity of the drugs was evaluated by CELLTITER-GLO®. Luminescentcell viability assay (Promega, Madison, Wis.). Pancreatic cell lineswere plated at a density of 2×10⁴/well in white 96 well, clear bottomplates for 24 hours prior to treatment. Drugs were dissolved in DMSO andserially diluted in culture medium to achieve the final concentration ofDMSO less than 1%. Cells were then treated for 24 hours and 100 μl ofthe CELLTITER-GLO® reagent was added to each well. The contents of theplates were mixed using an orbital shaker and subsequently incubated for10 minutes at room temperature. Luminescence signal was measured using amulti-mode microplate reader (BioTek instruments. Winooski, Vt.).Different drug concentrations were assayed in triplicate. To evaluatethe efficacy of the drugs, IC₅₀ of the compounds was calculated on apanel of human and mouse derived pancreatic cancer cell lines in vitro.Cells were treated for 24 hours with SW V-49s, SV119, Erastin, and anequimolar mixture of SV119 and Erastin, then CELLTITER-GLO® viabilityassay was performed. Erastin was the least active compound, withIC₅₀>150 μM. SV119 demonstrated a modest efficacy, that was augmented byadding Erastin. However, SW V-49s showed robust cytotoxicity with 17-20fold reduction of IC₅₀ as compared to treatment with the equimolarmixture of SV119 and Erastin (Table 4 and FIG. 9). These resultsindicate that SW V49s is selectively delivered to pancreatic cancercells.

TABLE 5 IC₅₀ (μM) of pancreatic cell lines treated with differentcompounds for 24 hours. Cell Line PANC-1 BxPC-3 AsPC-1 MiaPaCa-2 KCKODrugs IC₅₀ ± SEM IC₅₀ ± SEM IC₅₀ ± SEM IC₅₀ ± SEM IC₅₀ ± SEM SW V-49s 4.1 ± 0.2  2.5 ± 0.1  3.2 ± 0.3  3.0 ± 0.3 2.4 ± 0.2 SW119 + Erastin  70 ± 0.3 40.6 ± 2.5  46.5 ± 2.9 50.6 ± 2.5 48.4 ± 0.8  SV119 111.3 ±5.3 54.2 ± 2.6 111.3 ± 8.3 94.5 ± 1.9 68.7 ± 10.1Erastin >150 >150 >150 >150 >150 (Mean + SEM), n ≥ 3. P < 0.05

Since Erastin demonstrated no effects in all pancreatic cell lines, theinventors performed a quality control experiment using SYO-1 cells.AsPC-1 and SYO-1 cells were plated in 96 well plate overnight thentreated with similar concentrations of Erastin for 24 hours. Viabilityassay was performed after 24 hours. Treatment with 40 μM Erastinresulted in the death of 84% of SYO-1 cells, as compared to 2% of AsPC-1cells. (p<0.0001), (FIG. 10). This experiment demonstrates that theErastin used is bioactive and the pancreatic cell lines are resistant toErastin.

SW V-49s Inhibits Cystine Uptake and Generates Reactive Oxygen Species.

In these experiments, cystine uptake assay was performed as previouslydescribed (Dixon, S. J. et al. Cell 2012; 149: 1060-1072). Briefly,5×10^(51 AsPC-)1 cells/well were seeded overnight in 6 well plate. Thenext day, cells were washed twice in pre-warmed Na⁺-free uptake buffer(137 mM choline chloride, 3 mM KCl, 1 mM CaCl₂, 1 mM MgCl₂, 5 mMD-glucose, 0.7 mM K₂HPO₄, and 10 mM HEPES [pH 7.4]). Subsequently, cellswere incubated for 10 minutes at 37° C. in 1 ml of the uptake buffer todeplete cellular amino acids. The buffer was then replaced with 600 μluptake buffer containing 200 μM of SW V-49s and 0.12 μCi (80-110mCi/mmol) of L-[3,3′-¹⁴C]-cystine (American Radiolabeled Chemicals. StLouis. Mo.) and incubated for 3 minutes at 37° C. After that, cells werewashed three times with ice-cold uptake buffer and lysed in 500 μl of0.1 M NaOH. To this lysate, 1 ml of scintillation fluid was added, andradioactive counts per minute were obtained by using a scintillationcounter.

Erastin has been shown to block cystine uptake by inhibitingcystine/glutamate antiporter (system X_(c)′) resulting in ROS dependentcell death (Dixon et al. Cell 2012: 149: 1060-1072). To evaluate thismechanism. AsPC-1 cells were plated in 6 well plate for 24 hours. Cellswere then treated with the same concentration of SV119, Erastin,combination of SV119 and Erastin. SW V-49s, and DMSO as a control. Afterthat, a cystine uptake assay was performed. Cells treated with SW V-49sdemonstrated a reduction in cystine uptake by 85% (3.7 fold lesscysteine uptake) as compared to 25%, 41%, and 44% in the cells treatedwith SV 119. Erastin, and combination of SV 119 and Erastin,respectively (p=0.004, FIG. 11A).

ROS measurement was performed using Total ROS/Superoxide Detection Kit(Enzo life sciences, Farmingdale, N.Y.) according to the manufacturer'sinstructions. Briefly. As PC-1 cells were seeded at a density of 2×10⁴cells/well in a black wall clear bottom 96-well plate for 24 hours.Compounds were dissolved in DMSO and diluted in culture medium toachieve a final concentration of DMSO less than 1%. AsPC-1 cells weretreated with 8 μM of SV119, Erastin, and equimolar mixture of SV119 andErastin. ROS assay was performed one hour after treatment. Cells weretreated for one hour, then media was removed and 100 μL/well ofROS/Superoxide Detection Mix was added. Fluorescence signal was measuredusing a multi-mode microplate reader (Bio-Tek, Winooski, Vt.). The assaywas performed in 6 replicates.

The ROS level of the cells treated with SW V-49s was 1.5 fold highercompared to others. There was no increase in the ROS level in cellstreated with SV119. Erastin, and equimolar mixture of SV119 and Erastinat that concentration (FIG. 11B, p<0.0001).

Compound SW V-49s can Induce Intrinsic Apoptotic Pathway.

In these experiments, caspase-3/7.8 and 9 activities were measured InAsPC-1 cells using the corresponding CASPASE-GLO® Assay according to themanufacturer's instructions (Promega, Madison. Wis.). This assay isbased on a caspase-specific substrate, which is cleaved to releaseaminoluciferin, a substrate of luciferase that results incaspase-specific luminescence signals. Cells were seeded at a density of2×10⁴ in white 96-well, clear bottom plates for 24 hours beforetreatment with 4 μM of compounds. The contents were then mixed using aplate shaker for 30 seconds, thereafter incubated at room temperaturefor 90 minutes. Luminescence signal was measured using a multi-modemicroplate reader (BioTek). Assay was performed in triplicates, and thecaspase activity of DMSO was considered as a base line.

The present inventors have shown that SV119 induces caspase-3 dependentapoptosis (Kashiwagi, H., el al. Mol Cancer 2007; 6:48). In theseexperiments, AsPC-1 cells were treated with 4 μM of SW V-49s. SV119,Erastin, and equimolar mixture of SV119 and Erastin for 24 hours. UsingCASPASE-GLO® Assays, Caspase-3 level was measured to assess the activityof SW 119 domain of the SW V-49s compound and Caspase-8 and 9 levelswere measured to identify which apoptotic pathway was involved. Cellstreated with SW V-49s had a significant increase in caspase-3 and 9 (3.4and 3.2 fold above baseline respectively. ***p <0.001. FIG. 12A and FIG.12B, respectively). In contrast, there was no significant increase incaspase 8, ns >0.5 (FIG. 12C). Other compounds tested did not activateany of the caspases at a similar concentration (FIG. 12A-C). Theseresults suggest that SW V-49s can be selectively delivered to the cancercells and its two domains can work synergistically to activate theintrinsic apoptotic pathway.

SW V-49s can Induce ROS and Apoptotic Dependent Cell Death.

The present inventors demonstrated that SW V-49s can induce apoptosisand ROS generation. To assess the roles of these two mechanisms on theinduction of cell death, the effects of pan-caspase inhibitor andantioxidant on the efficacy of SW V-49s were tested. In theseexperiments, AsPC-1 cells were pre-treated with 10 mM of the antioxidantN acetyl cysteine (NAC), 20 μM of pan-caspase inhibitor ZVAD, and DMSOas a control for 1 hour. Then, the 3 groups were treated with 10 μM ofSW V-49s for 5 hours, after which a CELLTITER-GLO® viability assay wasperformed. The results indicated that viability of cells treated with SWV-49s alone was reduced to 39% compared to 63% and 91% in cellspretreated with NAC and ZVAD respectively (FIG. 13, p<0.0001). NAC wasfound to be more effective in inhibiting SW V-49s activity (FIG. 13,p<0.002). These data demonstrate the dual functionality of SW V-49s andindicate that it can induce both apoptotic and ROS dependent cell death.

SW V-49s Reduces Tumor Growth and Enhances Survival in Mouse and PatientDerived Xenograft Models of Pancreatic Cancer.

Animal studies were performed according to the animal studies protocolapproved by Washington University Institutional Animal Care Facility. Invivo studies with mice were performed to compare the effects of SWV-49s, SV119. Erastin, a combination of SV119 with Erastin, and vehicle.The vehicle used in the in vivo studies is a mixture of 25% cremophorand 75% H₂O. C57BL/6 mice (6 weeks old. National Cancer InstituteLaboratories) were injected in the right flank with 200 μL single cellsuspension of KCKO cells in RPMI medium (25×10⁴/10⁵ cells per mouse).Mice were randomized into four groups (n=15). Treatment was started whenthe mean tumor diameter was ^(˜)5 mm. Mice received daily intraperitoneal injections with 375 nmoles in 100 μL/dose of SW V-49s andvehicle for 10 days SV119. Erastin, equimolar mix of SV119 and Erastin.Tumors were measured every other day with a digital caliper. Severalmice from different treatment groups were sent to the Division ofComparative Medicine in our institution for pathologic evaluation. Bloodwas collected for complete blood count (CBC) and biochemical analysis(AST, ALT, BUN, total bilirubin, and Cr). Organs were examined grosslyand histologically.

In the syngeneic cancer model (KCKO in C57BL/6), only the SW V-49s wascapable of reducing the mean tumor volume (FIG. 14A, p=0.0003). None ofthe other reagents alone or in combination resulted in a reduction intumor growth, and they all had similar growth rates to the vehicle(control), (FIG. 14A, p=0.9). The median survival for the group treatedwith SW V49s was 48 days compared to (18-21) days for the other groups(FIG. 14B. Kaplan-Meier survival curve, p<0.001). Of note, no grossabnormalities in the mice behavior (grooming) or any drug-related deathswere recorded. This was supported by the unchanged serum labs (CBC, AST,ALT. BUN, total bilirubin, and Cr). (tables 5 and 6). In addition,organs analyses (brain, heart, lungs, alimentary tract, kidneys, liverand pancreas), did not reveal any obvious signs of adverse drug effects,except a mild peritonitis.

TABLE 6 SW V-49s Does Not Induce Changes in Blood Cytology (CBC)Following WBC RBC Platelets (10³/ (10⁶/ HGB PCV MCV MCH MCHC (10³/ IDμL) μL) (g/dl) (%) (u) (pg) (%) μL) Control 1 7.72 9.15 12.4 49.2 53.813.6 25.2 651 Control 2 8.10 8.89 11.2 45.6 51.3 12.6 24.6 745 Control 35.54 8.89 11.5 44.7 50.3 12.9 25.7 788 Drug 1 4.16 7.66 9.8 37.7 49.212.8 26.0 611 Drug 2 5.68 8.67 10.7 39.3 45.3 12.3 27.2 749 Drug 3 3.348.40 10.5 42.4 50.5 12.5 24.8 885

Table 6 contains data from CBC of C57BL/6 mice treated with SW V-49s andvehicle (control) for 10 days. The differences in cell counts betweenthe 2 groups are not statistically significant.

TABLE 7 SW V-49s Does Not Induce Changes In Serum Chemistry FollowingTreatment Of Tumor Bearing C57BL/6 Mice. Total BUN Creatinine ALT ASTGlucose Protein ID (mg/dL) (mg/dL) (μ/L) (μ/L) (mg/dL) (g/dL) Control 123 0.33 95 87 179 6.1 Control 2 17 0.2.6 65 73 180 5.4 Control 3 28 0.28506 715 187 5.5 Drug 1 19 0.22 138 167 178 5.1 Drug 2 22 0.21 64 147 3105.7 Drug 3 17 0.24 63 55 180 5.1

Table 7 shows biochemical analysis of C57BL/6 mice treated with SWIV-134 and vehicle (control) for 10 days. The differences in laboratoryvalues between the 2 groups are not statistically significant.

SW V-49s was also tested on a PDAC patient-derived mouse xenograftmodel, which is more clinically relevant. Surgical PDAC specimens (2×2mm pieces) were obtained and implanted subcutaneously into the flanks ofanesthetized NOD SCID mice. Then, tumors were harvested and implanted inthe right flank of Athymic female nude mice (6 weeks old. NationalCancer Institute Laboratories). These mice were treated with daily i.p.injections of SW IV-134 and vehicle for 14 days. Mice were randomizedinto 2 groups (n=15). Drug treatment was started when the mean tumorsdiameter was ^(˜)6 mm. Mice received daily intra peritoneal injectionswith 375 nmoles in 100 μL/mouse of SW V-49s and vehicle for 2 weeks.Tumors were measured every other day. Mice were euthanized when tumorsreached a diameter of 2 cm or ulcerated.

In this model, SW V-49s markedly slowed the growth rate of theestablished tumors (FIG. 14C. p<0.0001). The median survival for thegroup treated with SW V-49s was 58 days compared to 33 days for thevehicle group (FIG. 14D, Kaplan-Meier survival curve, p=0.0002). Micetolerated the treatment well without obvious off-target effects.

SYO-1 Synovial sarcoma (SS) xenografts were also treated with 300 nmolesSW V-49. Growth inhibition in a rapidly growing model of SS wasobserved. FIG. 14E depicts athymic nude mice xenograft of SS cell lineSYO-1 treated with SW V-49 (300 nmoles) for 14 days and then followed,p<0.0001. SYO xenografts required a smaller dose of SW V-49 to achieveresults similar to those achieved against PDAC. These resultsdemonstrate the high efficacy of SW V-49s in both pancreatic cancer andsynovial sarcoma and indicate its selective delivery.

Other Embodiments

The detailed description set-forth above is provided to aid thoseskilled in the art in practicing the present disclosure. However, thedisclosure described and claimed herein is not to be limited in scope bythe specific embodiments herein disclosed because these embodiments areintended as illustration of several aspects of the disclosure. Anyequivalent embodiments are intended to be within the scope of thisdisclosure. Indeed, various modifications of the disclosure in additionto those shown and described herein will become apparent to thoseskilled in the art from the foregoing description, which do not departfrom the spirit or scope of the present inventive discovery. Suchmodifications are also intended to fall within the scope of the appendedclaims.

All references cited are hereby incorporated by reference, each in itsentirety. Applicant reserves the right to challenge any conclusionspresented by the authors of any reference.

The invention claimed is:
 1. The methanesulfonate salt of a comnound of structural Formula TV.

wherein n is an integer chosen from 1, 2, 3, 4, and 5, and R₂ is H or methyl.
 2. The methanesulfonate salt of claim 1, wherein n is 1 and R₂ is H.
 3. The methanesulfonate salt of claim 1, wherein n is 5 and R₂ is H.
 4. A pharmaceutical composition comprising a compound of structural Formula IV

or a salt thereof and a pharmaceutically acceptable carrier, adjuvant, or vehicle.
 5. The pharmaceutical composition of claim 4, wherein the pharmaceutically acceptable carrier, adjuvant, or vehicle is water or saline.
 6. The pharmaceutical composition of claim 4 in the form of a solution.
 7. The pharmaceutical composition of claim 4, wherein the salt is methanesulfonate.
 8. The pharmaceutical composition of claim 4, formulated for oral delivery.
 9. The pharmaceutical composition of claim 4, formulated for intraperitoneal delivery.
 10. A method for treating cancer in a subject in need thereof, comprising administering to the subject in an effective amount of the pharmaceutical composition of claim 4, wherein the cancer is chosen from pancreatic cancer, synovial sarcoma, leukemia, lung cancer, non-small cell lung cancer, colorectal cancer, ovarian cancer, renal cell carcinoma, prostate cancer, and breast cancer.
 11. The method of claim 10, wherein the cancer is pancreatic cancer.
 12. The method of claim 10, wherein the cancer is synovial sarcoma.
 13. The method of claim 10, further comprising administering an additional anticancer drug to the subject.
 14. The method of claim 13, wherein the additional anticancer drug is an immunotherapy agent.
 15. The method of claim 13, wherein the additional anticancer drug is chosen from an alkylating agent, anthracycline, antimetabolite agent, crosslinking agent, D.N.A. replication inhibitor, intercalator, microtubule disruptor, PARP inhibitor, radiomimetic agent, radiosensitizer, strand break agent, and topoisomerase II inhibitor.
 16. The method of claim 13, wherein the additional anticancer drug is chosen from aminoglutethimide, amsacrine, anastrozole, asparaginase, barasertib, beg, bicalutamide, bleomycin, buserelin, busulfan, campothecin, capecitabine, carboplatin, carmustine, chlorambucil, chloroquine, cisplatin, cladribine, clodronate, colchicine, cyclophosphamide, cyproterone, cytarabine, dacarbazine, dactinomycin, daunorubicin, demethoxyviridin, dichloroacetate, dienestrol, diethylstilbestrol, docetaxel, doxorubicin, epirubicin, estradiol, estramustine, etoposide, everolimus, exemestane, filgrastim, fludarabine, fludrocortisone, fluorouracil, fluoxymesterone, flutamide, gemcitabine, genistein, goserelin, hydroxyurea, idarubicin, ifosfamide, imatinib, interferon, irinotecan, ironotecan, letrozole, leucovorin, leuprolide, levamisole, lomustine, lonidamine, mechlorethamine, medroxyprogesterone, megestrol, melphalan, mercaptopurine, mesna, metformin, methotrexate, mitomycin, mitotane, mitoxantrone, nilutamide, nocodazole, olaparib, octreotide, oxaliplatin, paclitaxel, pamidronate, pentostatin, perifosine, plicamycin, porfimer, procarbazine, raltitrexed, rituximab, sorafenib, streptozocin, sunitinib, suramin, tamoxifen, temozolomide, temsirolimus, teniposide, testosterone, thioguanine, thiotepa, titanocene dichloride, topotecan, trastuzumab, tretinoin, vinblastine, vincristine, vindesine, and vinorelbine.
 17. The method of claim 13, wherein the method further comprises administering radiation therapy.
 18. The method of claim 13, further comprising administering surgery, thermoablation, focused ultrasound therapy, cryotherapy, or any combination thereof. 